Compositions and methods for the treatment of immunologic disorders

ABSTRACT

Compositions and methods for preventing, reducing or inhibiting immunologic disorders are provided. Suitable compositions include one or more LIGHT-HVEM antagonists. LIGHT-HVEM antagonists include compounds that inhibit, reduce, or block the biological activity or expression of LIGHT and/or HVEM. LIGHT-HVEM antagonists can reduce or inhibit the binding of LIGHT to HVEM, but do not significantly modulate the binding of LTβ to LTβR. Suitable compositions include antibodies and antibody fragments, decoy polypeptides, small molecule inhibitors and inhibitory nucleic acids. Methods for using LIGHT-HVEM antagonists to reduce or inhibit T cell activation and survival are also provided. Therapeutic uses for LIGHT-HVEM antagonists to prevent or treat immunologic diseases and disorders including graft rejection, graft-versus-host disease, inflammatory immune responses, and autoimmune disorders are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of Provisional U.S.Patent Application No. 60/877,176 filed on Dec. 26, 2006.

GOVERNMENT SUPPORT

This invention was made with government support awarded by the NationalInstitutes of Health under Grant Number CA 1085721. The United Statesgovernment has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to compositions and methods for reducinginflammatory immune responses, in particular to compositions and methodsfor treating or inhibiting inflammatory responses related to autoimmunedisorders or immune responses related to transplanted cells or tissuesincluding graft-versus-host disease.

BACKGROUND OF THE INVENTION

The functional network of tumor necrosis factor (TNF) and TNF receptorsuperfamily members is composed of complex cross-talk between multipleligands and multiple receptors, which regulate pleiotropic functions inthe immune system (Aggarwal, Nat. Rev. Immunol., 3(9):745-56 (2003)).LIGHT, standing for homologous to lymphotoxins, exhibits inducibleexpression, and competes with herpes simplex virus glycoprotein D forherpesvirus entry mediator (HVEM), a receptor expressed by Tlymphocytes, is a type II transmembrane glycoprotein belonging to theTNF ligand superfamily (Mauri, et al., Immunity, 8(1):21-30 (1998)).LIGHT is expressed on immature dendritic cells (DCs) and activated Tcells (Mauri, et al., Immunity, 8(1):21-30 (1998); Tamada, et al., J.Immunol., 164(8):4105-10 (2000)) and interacts with two functionalreceptors: lymphotoxin-β receptor (LTβR) and HVEM (Mauri, et al.,Immunity, 8(1):21-30 (1998)). LIGHT interaction with LTβR triggers theproduction of proinflammatory mediators (Lee, et al., Arterioscler.Thromb. Vasc. Biol., 21(12):2004-10 (2001); Kim, et al., Immunology,114(2):272-9 (2005)), up-regulates adhesion molecule expression (Yu, etal., Nat. Immunol., 5(2):141-9 (2004)), and induces apoptotic cell deathin certain tumors (Rooney, et al., J. Biol. Chem., 275(19):14307-15(2000)). On the other hand, by signaling through HVEM, LIGHTcostimulates T-cell activation (Tamada, et al., Nat. Med., 6(3):283-9(2000)). In vivo experiments demonstrated that transgenic expression ofLIGHT leads to spontaneous progression of inflammatory autoimmunity suchas Crohn's disease (Wang, et al., J. Clin. Invest., 108(12):1771-80(2001); Shaikh, et al., J. Immunol., 167(11):6330-7 (2001); Wang, etal., J. Immunol., 174(12):8173-82 (2005)), while genetic disruption ofLIGHT results in impaired T-cell activation, particularly in CD8⁺ Tcells (Ye, et al., J. Exp. Med., 195:795-800 (2002); Tamada, et al., J.Immunol., 168:4832-4835 (2002); Scheu, et al., J. Exp. Med.,195:1613-1624 (2002); Liu, et al., Int. Immunol., 15:861-870 (2003)),and renders mice less vulnerable to pathogenic inflammation, as shown inacute hepatitis models (Anand, et al., J. Clin. Invest, 116:1045-1051(2006)). Thus, LIGHT regulates multiple immune functions of innate andadaptive immunity through interactions with LTβR and HVEM.

There are reports demonstrating therapeutic effects of decoy proteins ofLTβR in various immunologic diseases, including autoimmunity,inflammation, and transplantation (Gommerman, et al., Nat. Rev.Immunol., 3:642-655 (2003); Spahn, et al., Infect. Immun., 73:7077-7088(2005)), indicating that decoy LTβR could be a potential biologic forclinical immunotherapy, analogous to a decoy form of TNF-receptor(Moreland, et al., N. Engl. J. Med., 337:141-147 (1997)). Prolongedadministration of decoy LTβR, however, might become a double-edged swordsince it abrogates the maintenance of DC and natural killer/naturalkiller T (NK/NKT) cells (Wu, et al., J. Exp. Med., 190:629-638 (1999);Iizuka, et al., Proc. Natl. Acad. Sci. U.S.A., 96:6336-6340 (1999)) andinhibits the microstructure formation of lymphoid organs (Mackay, etal., Eur. J. Immunol., 27:2033-2042 (1997)), thus disrupting immunehomeostasis.

Graft-versus-host disease (GVHD) is a major complication associated withallogeneic hematopoietic stem cell transplantation. Posttransplantationadministration of immunosuppressants prevails as the current therapeuticchoice for GVHD, but this treatment results in systemicimmunosuppression that often leads to opportunistic pathogen infectionsand leukemic relapse (Murphy, et al, Curr. Opin. Immunol., 11:509-515(1999); Blazar, et al., Philos. Trans. R. Soc. Lond. B Biol. Sci.,360:1747-1767 (2005)). To overcome these issues, blockade of T-cellcostimulatory signals is among the most sought after alternatives toimmunosuppressants (Murphy, et al., Curr. Opin. Immunol., 11:509-515(1999); Blazar, et al., Philos. Trans. R. Soc. Lond. B Biol. Sci.,360:1747-1767 (2005)). Previous findings have suggested a therapeuticpotential of LIGHT costimulation, in which administration of LTβR-Ig, adecoy LTβR, inhibits alloreactive cytotoxic T lymphocyte (CTL)generation and prolongs the survival of GVHD mice (Tamada, et al., Nat.Med., 6:283-289 (2000)). Combined therapy of LTβR-Ig and anti-CD40ligand monoclonal antibody (mAb) further protects the recipient micefrom GVHD by rendering alloreactive donor CTL anergic (Tamada, et al.,J. Clin. Invest., 109:549-557 (2002)). However, the actual contributionof the LIGHT-HVEM costimulatory system to these findings remains elusivedue to the antihomeostatic effects of decoy LTβR. It is possible thatchanges of DC function or cellular structure in lymphoid tissues couldaffect the intensity of adaptive immune responses. Direct evidenceindicating a pathogenic role of LIGHT-HVEM costimulation in GVHD has notbeen elucidated.

It would be advantageous to provide new compositions and methods forseparating the therapeutic effects of decoy LTβR from the potentialadverse effects. While decoy LTβR interferes with three molecularinteractions—LTβ-LTβR, LIGHT-LTβR, and LIGHT-HVEM—the antihomeostaticeffects are largely dependent on LTβ-LTβR functions since thecorresponding phenotypes are observed in LTβ- or LTβR-KO mice but not inLIGHT-KO mice (Ye, et al., J. Exp. Med., 195:795-800 (2002); Tamada, etal., J. Immunol., 168:4832-4835 (2002); Scheu, et al., J. Exp. Med.,195:1613-1624 (2002); Liu, et al., Int. Immunol., 15:861-870 (2003);Kabashima, et al., Immunity, 22:439-450 (2005); Wu, et al., J. Immunol.,166:1684-1689 (2001); Alimzhanov, et al., Proc. Natl. Acad. Sci. U.S.A.,94:9302-9307 (1997); Futterer, et al., Immunity, 9:59-70 (1998)).

Therefore, it is an object of the invention to provide compositions andmethods of use thereof that reduce inflammatory immune responses but donot significantly disrupt normal immune system homeostasis such as byinterfering with the maintenance of DC and natural killer/natural killerT (NK/NKT) cells or by inhibiting the microstructure formation oflymphoid organs.

It is another object of the invention to provide compositions andmethods of use thereof that inhibit or reduce T cell costimulatorysignals.

It is another object of the invention to provide compositions andmethods for the treatment of immunologic disorders, includinginflammatory responses.

It is another object of the invention to provide compositions andmethods for the treatment of autoimmune disorders.

It is still another object of the invention to provide compositions andmethods for treatment of graft rejection and graft-versus-host disease.

SUMMARY OF THE INVENTION

Compositions and methods for preventing, reducing or inhibitingimmunologic disorders are provided herein. Suitable compositions includeone or more LIGHT-HVEM antagonists. LIGHT-HVEM antagonists includecompounds that inhibit, reduce, or block the biological activity orexpression of LIGHT and/or HVEM.

LIGHT-HVEM antagonists can reduce or inhibit the binding of LIGHT toHVEM, but do not significantly modulate the binding of LTβ to LTβR.Exemplary LIGHT-HVEM antagonists that reduce or inhibit the binding ofLIGHT to HVEM include antibodies and antigen-binding antibody fragments,decoy polypeptides and small molecule inhibitors. LIGHT-HVEM antagoniststhat are capable of binding to LIGHT or HVEM do not increase LIGHT orHVEM activity in a cell expressing LIGHT or HVEM on its surface. In someembodiments LIGHT-HVEM antagonists are capable of reducing or inhibitingone or more activities of LIGHT or HVEM in a cell expressing LIGHT orHVEM on its surface. In some embodiments, the cell is a lymphocyte, a Tcell, a CD4+ T cell, a CD8+ T cell, a T_(h)1 cell, a B cell, a plasmacell, a macrophage, or an NK cell. In preferred embodiments, the cell isa T cell.

LIGHT-HVEM antagonistic antibodies bind to LIGHT or HVEM and reduce orinhibit the binding of LIGHT to HVEM. In preferred embodiments,LIGHT-HVEM antagonistic antibodies specifically bind to an extracellularportion of LIGHT or HVEM. LIGHT-HVEM antagonistic antibodies can bemonoclonal or polyclonal, antiidiotypic, xenogeneic, allogeneic,syngeneic, or modified forms thereof, such as humanized or chimericantibodies. The antibodies can also be antibody fragments or singlechain antibodies. An exemplary antibody is mAb LBH1.

Polypeptides that bind to LIGHT or HVEM and inhibit or reduce bindingbetween LIGHT and HVEM are also provided. Suitable polypeptides includefragments of LIGHT or HVEM. In preferred embodiments, the polypeptidesare soluble fragments of LIGHT or HVEM. Exemplary soluble fragments ofLIGHT and HVEM include the extracellular domains of LIGHT or HVEM orfragments thereof. Other suitable polypeptides include soluble fragmentsor receptors of HVEM that are not LIGHT and soluble fragments orreceptors of LIGHT that are not HVEM. Exemplary of these polypeptidesare lymphotoxin-α, herpesvirus gD protein and the TR6 receptor.Polypeptide LIGHT-HVEM antagonists may contain one or moresubstitutions, deletions or insertions of amino acids relative to theirwild-type sequence, and may be in the form of fusion proteins.

LIGHT-HVEM antagonists that reduce or inhibit the expression of LIGHT orHVEM are also provided. Suitable LIGHT-HVEM antagonists includeinhibitory nucleic acids, including, but not limited to, ribozymes,triplex-forming oligonucleotides (TFOs), antisense DNA, siRNA, andmicroRNA specific for nucleic acids encoding LIGHT or HVEM.

Methods for using LIGHT-HVEM antagonists to reduce or inhibit T cellactivation and survival are also provided. Therapeutic uses forLIGHT-HVEM antagonists are provided. LIGHT-HVEM antagonists can be usedto prevent or treat immunologic diseases and disorders including graftrejection, graft-versus-host disease, inflammatory immune responses, andautoimmune disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a series of line graphs showing anti-host cytotoxic Tlymphocyte (CTL) activity of spleen cells isolated from recipient BDF1(H-2^(d)) mice intravenously injected with either wild-type (WT) orLIGHT-KO B6 (H-2^(b)) spleen cells. CTL activity was measured againstP815 (H-2^(d)) and EL4 (H-2^(b)) tumor cells by ⁵¹Cr-release assay. Dataare expressed as percent lysis as a function of effector cell/targetcell ratio. The closed squares represent data obtained using spleencells from BDF1 mice injected with spleen cells from WT B6 mice and theopen squares represent data obtained using spleen cells from BDF1 miceinjected with spleen cells from LIGHT-KO B6 mice.

FIG. 1B is a series of line graphs showing anti-host cytotoxic Tlymphocyte (CTL) activity of spleen cells isolated from recipient BDF1(H-2^(d)) mice intravenously injected with various combinations of Tcells and non-T cells from either wild-type (WT) or LIGHT-KO B6(H-2^(b)) mice. CTL activity was measured against P815 (H-2^(d)) and EL4(H-2^(b)) tumor cells by ⁵¹Cr-release assay. Data are expressed aspercent lysis as a function of effector cell/target cell ratio. T cellsand non-T cells purified from spleen cells of WT or LIGHT-KO B6 micewere injected in BDF1 mice in the following combinations: WT T cellsplus WT non-T cells (open squares), WT T cells plus LIGHT-KO non-T cells(closed squares), LIGHT-KO T cells plus WT non-T cells (open circles),and LIGHT-KO T cells plus LIGHT-KO non-T cells (closed circles).

FIG. 1C is a line graph showing survival of BDF1 mice subjected tolethal-dose irradiation (12 Gy) followed by intravenous injection of Tcell-depleted B6 BM cells alone (open circles) or together with WT(closed squares) or LIGHT-KO (closed triangles) B6 T cells. Data areexpressed as percent survival as a function of time in days.

FIG. 1D is a series of line graphs showing survival and body weightchanges of BALB/c mice subjected to lethal-dose irradiation (10 Gy)followed by intravenous injection of T cell-depleted B6 BM cells alone(closed squares) or together with WT (closed circles) or LIGHT-KO (opencircles) B6 T cells. Data are expressed as percent survival or percentbody weight as a function of time in days.

FIG. 2 is a series of bar graphs showing absolute numbers of WT (closedbars) or LIGHT-KO (open bars) B6 donor CD4⁺ or CD8⁺ T cells in totalspleen or liver lymphocytes from recipient BDF1 mice. Data are expressedas total cell number (×10⁶).

FIG. 3A is a series of histograms showing proliferation of WT orLIGHT-KO B6 donor CD4+ or CD8+ T cells from spleens of recipient BDF1mice. Proliferation was measured by intensity of CFSE staining ofH-2K^(d)-negative, CD4⁺ or CD8⁺ cells at days 2, 4 and 6 after injectionof donor cells into BDF1 mice. The percentage of donor T cells with morethan one division is indicated in each panel.

FIG. 3B is a series of histograms showing apoptosis of WT or LIGHT-KO B6donor CD4⁺ or CD8⁺ T cells from spleens of recipient BDF1 mice.Apoptosis was measured by the intensity of Annexin V staining 7 daysafter injection of donor cells into BDF1 mice. The percentage of AnnexinV-positive donor T cells is indicated in each panel.

FIG. 4A is a series of line graphs showing anti-host cytotoxic Tlymphocyte (CTL) activity of spleen cells isolated from recipient BDF1(H-2^(d)) mice intravenously injected with either wild-type (WT) orHVEM-KO B6 (H-2^(b)) spleen cells. CTL activity was measured againstP815 (H-2^(d)) and EL4 (H-2^(b)) tumor cells by ⁵¹Cr-release assay. Dataare expressed as percent lysis as a function of effector cell/targetcell ratio. The closed squares represent data obtained using spleencells from BDF1 mice injected with spleen cells from WT B6 mice and theopen squares represent data obtained using spleen cells from BDF1 miceinjected with spleen cells from HVEM-KO B6 mice.

FIG. 4B is a series of line graphs showing anti-host cytotoxic Tlymphocyte (CTL) activity of spleen cells isolated from recipient BDF1(H-2^(d)) mice intravenously injected with various combinations of Tcells and non-T cells from either wild-type (WT) or HVEM-KO B6 (H-2^(b))mice. CTL activity was measured against P815 (H-2^(d)) and EL4 (H-2^(b))tumor cells by ⁵¹Cr-release assay. Data are expressed as percent lysisas a function of effector cell/target cell ratio, T cells and non-Tcells purified from spleen cells of WT or HVEM-KO B6 mice were injectedin BDF1 mice in the following combinations: WT T cells plus WT non-Tcells (open squares), WT T cells plus HVEM-KO non-T cells (closedsquares), HVEM-KO T cells plus WT non-T cells (open circles), andHVEM-KO T cells plus HVEM-KO non-T cells (closed circles).

FIG. 4C is a series of histograms showing apoptosis of WT or HVEM-KO B6donor CD4⁺ or CD8⁺ T cells from spleens of recipient BDF1 mice.Apoptosis was measured by the intensity of Annexin V staining 7 daysafter injection of donor cells into BDF1 mice. The percentage of AnnexinV-positive donor T cells is indicated in each panel.

FIG. 4D is a series of line graphs showing survival of BALB/c micesubjected to lethal-dose irradiation (10 Gy) followed by intravenousinjection of T cell-depleted B6 BM cells alone (closed circles) ortogether with WT (closed squares) or HVEM-KO (open circles) B6 T cells.Data are expressed as percent survival as a function of time in days.

FIG. 5A is a series of histograms showing binding characteristics of theLBH1 mAb. 293T cells transfected with either full-length mouse HVEM(filled histogram) or control vector (open histogram) were stained withLBH1 (right) or control hamster IgG (left), followed by PE-conjugatedanti-hamster IgG Ab. Human 293T transfected with full-length mouse LIGHT(middle) or mouse BTLA (bottom) were stained with mouse HVEM-mouse Igfusion protein (filled histogram) or control mouse Ig protein (openhistogram) in the presence of 10 μg/mL LBH1 (right) or control hamsterIgG (left). Staining intensity of fusion protein was detected byFITC-conjugated anti-mouse Ig Ab.

FIG. 5B is a line graph showing the effect of the LBH1 mAb on T cellcostimulation. Tissue culture plates were coated with graded doses ofanti-CD3 mAb and further with LBH1 (closed triangles) or control hamsterIgG (open squares). Purified B6 T cells were cultured in the wells for 3days, and the proliferation activity during the last 15 hours wasmeasured by ³H-thymidine incorporation assay. As positive control, 3μg/mL anti-CD28 mAb was added in the wells at the beginning of T-cellculture (open circles).

FIG. 6A is line graph showing survival of BDF1 mice subjected tolethal-dose irradiation (12 Gy) followed by intravenous injection of Tcell-depleted B6 BM cells alone (open circles) or together with WT B6 Tcells. In the groups receiving T cell transfer, the mice were treatedintraperitoneally with LBH1 mAb (closed circles) or control hamster IgG(open squares) on days 0, 3 and 6 after BM transfer. Data are expressedas percent survival as a function of time in days.

FIG. 6B is a line graph showing survival of B6 mice subjected tolethal-dose irradiation (10 Gy) followed by intravenous injection of Tcell-depleted CH3.SW BM cells alone (open circles) or together withCH3.SW spleen cells. In the groups receiving spleen cell transfer, themice were treated intraperitoneally with LBH1 mAb (closed triangles) orcontrol hamster IgG (closed squares) on days 0, 5, 10, 15, 20 and 25after BM transfer. Data are expressed as percent survival as a functionof time in days.

FIG. 6C is a series of bar graphs showing change in body weight or GVHDclinical score. B6 mice were subjected to lethal-dose irradiation (10Gy) followed by intravenous injection of T cell-depleted CH3.SW BM cellstogether with CH3.SW spleen cells. The mice were treatedintraperitoneally with LBH1 mAb (open bar) or control hamster IgG(filled bar) on days 0, 5, 10, 15, 20 and 25 after BM transfer. Data areexpressed as average±standard deviation.

FIG. 7A is a series of line graphs showing anti-host cytotoxic Tlymphocyte (CTL) activity of spleen cells isolated from recipient BDF1(H-2^(d)) mice intravenously injected with WT B6 (H-2^(b)) spleen cells.Mice were treated with intraperitoneal administration of control hamsterIgG (closed squares) or LBH1 mAb on days 0, 3 and 6 after spleen celltransfer. CTL activity was measured on day 10 against P815 (H-2^(d)) andEL4 (H-2^(b)) tumor cells by ⁵¹Cr-release assay. Data are expressed aspercent lysis as a function of effector cell/target cell ratio.

FIG. 7B is a series of histograrns showing proliferation of CFSE-labeledB6 donor CD4+ or CD8+ T cells from spleens of recipient BDF1 micetreated with intraperitoneal injections of either control hamster IgG orLBH1 anti-HVEM antibody on days 0 and 3. Proliferation was measured byintensity of CFSE staining of H-2K^(d)-negative, CD4⁺ or CD8⁺ cells atdays 4 after injection of donor cells into BDF1 mice. The percentage ofdonor T cells with more than one division is indicated in each panel.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Terms defined herein have meanings as commonly understood by a person ofordinary skill in the art Terms such as “a”, “an” and “the” are notintended to refer to only a singular entity, but include the generalclass of which a specific example may be used for illustration.

As used herein, the term “LIGHT-HVEM antagonist” refers to compoundsthat inhibit, reduce, or block the biological activity or expression ofLIGHT and/or HVEM. LIGHT-HVEM antagonists disclosed herein do notsignificantly modulate the binding of LTβ to LTβR. Suitable LIGHT-HVEMantagonists include, but are not limited to, antibodies and antibodyfragments that bind HVEM or LIGHT, LIGHT or HVEM decoy polypeptidesincluding soluble fragments of LIGHT or HVEM, small organic compounds,and inhibitory nucleic acids specific for LIGHT- or HVEM-encodingnucleic acids.

As used herein the term “isolated” is meant to describe a compound ofinterest (e.g., either a polynucleotide or a polypeptide) that is in anenvironment different from that in which the compound naturally occurse.g. separated from its natural milieu such as by concentrating apeptide to a concentration at which it is not found in nature.“Isolated” is meant to include compounds that are within samples thatare substantially enriched for the compound of interest and/or in whichthe compound of interest is partially or substantially purified.

As used herein, the term “polypeptide” refers to a chain of amino acidsof any length, regardless of modification (e.g., phosphorylation orglycosylation).

As used herein, a “variant” polypeptide contains at least one amino acidsequence alteration (addition, deletion, substitution, preferablyconservative i.e., not substantially changing the function except inmagnitude) as compared to the amino acid sequence of the correspondingwild-type polypeptide.

As used herein, an “amino acid sequence alteration” can be, for example,a substitution, a deletion, or an insertion of one or more amino acids.

As used herein, a “vector” is a replicon, such as a plasmid, phage, orcosmid, into which another DNA segment may be inserted so as to bringabout the replication of the inserted segment. The vectors describedherein can be expression vectors.

As used herein, an “expression vector” is a vector that includes one ormore expression control sequences

As used herein, an “expression control sequence” is a DNA sequence thatcontrols and regulates the transcription and/or translation of anotherDNA sequence.

As used herein, “operably linked” means incorporated into a geneticconstruct so that expression control sequences effectively controlexpression of a coding sequence of interest.

As used herein, a “fragment” of a polypeptide refers to any subset ofthe polypeptide that is a shorter polypeptide of the full lengthprotein. Generally, fragments will be five or more amino acids inlength.

As used herein, “conservative” amino acid substitutions aresubstitutions wherein the substituted amino acid has similar structuralor chemical properties.

As used herein, “non-conservative” amino acid substitutions are those inwhich the charge, hydrophobicity, or bulk of the substituted amino acidis significantly altered.

As used herein, “isolated nucleic acid” refers to a nucleic acid that isseparated from other nucleic acid molecules that are present in amammalian genome, including nucleic acids that normally flank one orboth sides of the nucleic acid in a mammalian genome.

As used herein with respect to nucleic acids, the term “isolated”includes any non-naturally-occurring nucleic acid sequence, since suchnon-naturally-occurring sequences are not found in nature and do nothave immediately contiguous sequences in a naturally-occurring genome.

As used herein, the term “host cell” refers to prokaryotic andeukaryotic cells into which a recombinant expression vector can beintroduced.

As used herein, “transformed” and “transfected” encompass theintroduction of a nucleic acid (e.g. a vector) into a cell by a numberof techniques known in the art.

The terms “individual”, “host”, “subject”, and “patient” are usedinterchangeably herein.

As used herein the term “effective amount” or “therapeutically effectiveamount” means a dosage sufficient to treat, inhibit, or alleviate one ormore symptoms of an inflammatory response or autoimmune disease statebeing treated or to otherwise provide a desired pharmacologic and/orphysiologic effect. The precise dosage will vary according to a varietyof factors such as subject-dependent variables (e.g., age, immune systemhealth, etc.), the disease, and the treatment being effected.

As used herein, the phrase that a molecule “specifically binds” to atarget refers to a binding reaction which is determinative of thepresence of the molecule in the presence of a heterogeneous populationof other biologics. Thus, under designated immunoassay conditions, aspecified molecule binds preferentially to a particular target and doesnot bind in a significant amount to other biologics present in thesample. Specific binding of an antibody to a target under suchconditions requires the antibody be selected for its specificity to thetarget. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. See,e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, ColdSpring Harbor Publications, New York, for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity. Specific binding between two entities means anaffinity of at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. Affinities greaterthan 10⁸ M⁻¹ are preferred.

As used herein, the terms “antibody” or “immunoglobulin” are used toinclude intact antibodies and binding fragments thereof. Typically,fragments compete with the intact antibody from which they were derivedfor specific binding to an antigen fragment including separate heavychains, light chains Fab, Fab′ F(ab′)2, Fabc, and Fv. Fragments areproduced by recombinant DNA techniques, or by enzymatic or chemicalseparation of intact immunoglobulins. The term “antibody” also includesone or more immunoglobulin chains that are chemically conjugated to, orexpressed as, fusion proteins with other proteins. The term “antibody”also includes bispecific antibody. A bispecific or bifunctional antibodyis an artificial hybrid antibody having two different heavy/light chainpairs and two different binding sites. Bispecific antibodies can beproduced by a variety of methods including fusion of hybridomas orlinking of Fab′ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp.Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553(1992).

As used herein, an “antigen” is an entity to which an antibodyspecifically binds.

As used herein, the terms “epitope” or “antigenic determinant” refer toa site on an antigen to which B and/or T cells respond. B-cell epitopescan be formed both from contiguous amino acids or noncontiguous aminoacids juxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, and more usually, at least 5 or 8-10 amino acids, in a uniquespatial conformation. Methods of determining spatial conformation ofepitopes include, for example, x-ray crystallography and 2-dimensionalnuclear magnetic resonance. See, e.g., Epitope Mapping Protocols inMethods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).Antibodies that recognize the same epitope can be identified in a simpleimmunoassay showing the ability of one antibody to block the binding ofanother antibody to a target antigen. T-cells recognize continuousepitopes of about nine amino acids for CD8 cells or about 13-15 aminoacids for CD4 cells. T cells that recognize the epitope can beidentified by in vitro assays that measure antigen-dependentproliferation, as determined by ³H-thymidine incorporation by primed Tcells in response to an epitope (Burke et al., J. Inf. Dis. 170, 1110-19(1994)), by antigen-dependent killing (cytotoxic T lymphocyte assay,Tigges et al., J. Immunol. 156, 3901-3910) or by cytokine secretion.

As used herein, the terms “immunologic”, “immunological” or “immune”response is the development of a beneficial humoral (antibody mediated)and/or a cellular (mediated by antigen-specific T cells or theirsecretion products) response directed against an amyloid peptide in arecipient patient. Such a response can be an active response induced byadministration of immunogen or a passive response induced byadministration of antibody or primed T-cells. A cellular immune responseis elicited by the presentation of polypeptide epitopes in associationwith Class I or Class II MHC molecules to activate antigen-specific CD4⁺T helper cells and/or CD8⁺ cytotoxic T cells. The response may alsoinvolve activation of monocytes, macrophages, NK cells, basophils,dendritic cells, astrocytes, microglia cells, eosinophils or othercomponents of innate immunity. The presence of a cell-mediatedimmunological response can be determined by proliferation assays (CD4⁺ Tcells) or CTL (cytotoxic T lymphocyte) assays. The relativecontributions of humoral and cellular responses to the protective ortherapeutic effect of an immunogen can be distinguished by separatelyisolating antibodies and T-cells from an immunized syngeneic animal andmeasuring protective or therapeutic effect in a second subject.

An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant.

II. Compositions

A. LIGHT-HVEM Antagonists

It has been discovered that LIGHT and HVEM promote T cell activation andsurvival during immunologic responses. It is believed that LIGHT-HVEMbinding on T cells is the mechanism for promoting activation andsurvival. Compositions including on or more LIGHT-HVEM antagonists areprovided herein. LIGHT-HVEM antagonists include compounds that inhibit,reduce, or block the biological activity or expression of LIGHT and/orHVEM.

In certain embodiments, the compositions include as an active agent oneor more LIGHT-HVEM antagonists in an amount effective to inhibit,reduce, or decrease an immunologic response.

Human LIGHT is expressed as at least two isoforms produced throughalternative splicing of LIGHT mRNA. The amino acid sequences of humanLIGHT isoforms are known in the art and are provided at GENBANKaccession numbers NP_(—)003798 (isoform1) and NP_(—)742011 (isoform 2).The nucleic acid sequence of human LIGHT isoform mRNAs are known in theart and are provided at GENBANK accession numbers NM_(—)003807(isoform 1) and NM_(—)172014 (isoform 2). The coding region of the LIGHTmRNA provided by accession number NM_(—)172014 is from nucleotides383-997. The coding region of the LIGHT mRNA provided by accessionnumber NM_(—)003807 is from nucleotides 383-1105.

The amino acid sequence of human HVEM is known in the art and isprovided at GENBANK accession number AAQ89238. Full length human HVEM is283 amino acids in length. The nucleic acid sequence of human HVEM mRNAis known in the art and is provided at GENBANK accession numberAY358879. The coding region of the HVEM mRNA is from nucleotides 82 to933 of accession number AY358879.

Both LIGHT and HVEM are expressed as transmembrane proteins, each withan intracellular domain, a single membrane-spanning domain, and anextracellular domain. The extracellular domain of human LIGHT includesfrom about amino acid 60 to amino acid 240 of GENBANK accession numberNP_(—)003798 and from about amino acid to amino acid of GENBANKaccession number NP_(—)742011 (NP_(—)742011 variant lacks transmembraneregion and probably soluble protein). The extracellular domain of humanHVEM includes from about amino acid 1 to amino acid 202 of GENBANKaccession number AAQ89238.

The extracellular domain of LIGHT includes β-strand scaffold forming ananti-parallel β-sandwich structure and assembling a trimer.Extracellular domain of LIGHT has a single potential site of N-linkedglycan. The extracellular domain of HVEM includes four cysteine-richrepeats and two potential sites of N-linked glycans. HVEM binds withLIGHT through second and third cysteine-rich domains.

1. LIGHT-HVEM Antagonists that Reduce or Inhibit the Binding of LIGHT toHVEM

LIGHT-HVEM antagonists that reduce or inhibit the binding of LIGHT toHVEM do not significantly modulate the binding of LTβ to LTβR. Inpreferred embodiments LIGHT-HVEM antagonists reduce the binding of LTβto LTβR by less than 50%, 40%, 30%, 20%, 10%, 5% or less, as compared tocontrols. LIGHT-HVEM antagonists can be competitive or non-competitiveinhibitors of LIGHT-HVEM binding. LIGHT-HVEM antagonists that reduce orinhibit the binding of LIGHT to HVEM include antibodies and antibodyfragments that bind HVEM or LIGHT, LIGHT or HVEM decoy polypeptidesincluding soluble fragments of LIGHT or HVEM, and small organiccompounds.

LIGHT-HVEM antagonists that bind to LIGHT or HVEM reduce or inhibit theinteraction between LIGHT and HVEM by at least 20%, more preferably byat least 30%, more preferably by at least 40%, 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99% or more.

LIGHT-HVEM antagonists that are capable of binding to LIGHT or HVEM donot increase LIGHT or HVEM activity in a cell expressing LIGHT or HVEMon its surface. In some embodiments LIGHT-HVEM antagonists are capableof reducing or inhibiting one or more activities of LIGHT or HVEM in acell expressing LIGHT or HVEM on its surface. In some embodiments, thecell is a lymphocyte, a T cell, a CD4+ T cell, a CD8+ T cell, a T_(h)1cell, a B cell, a plasma cell, a macrophage, or an NK cell. In preferredembodiments, the cell is a T cell. LIGHT-HVEM antagonists that bind toLIGHT or HVEM reduce or inhibit one or more LIGHT or HVEM activities byat least 20%, more preferably by at least 30%, more preferably by atleast 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more.

a. Antibodies

In one embodiment, LIGHT-HVEM antagonists are antibodies. Antibodies orantibody fragments that specifically bind to LIGHT or HVEM can be usedto reduce or inhibit the binding of LIGHT to HVEM. Methods of producingantibodies are well known and within the ability of one of ordinaryskill in the art and are described in more detail below.

The antibodies disclosed herein specifically bind to a LIGHT or an HVEMprotein and are capable of reducing or inhibiting the binding of LIGHTto HVEM. These antibodies are defined as “blocking”, “function-blocking”or “antagonistic” antibodies. In preferred embodiments the antagonisticantibodies specifically bind to a portion of the extracellular domain ofLIGHT or HVEM. In other embodiments, the antagonistic antibodiesspecifically bind to the β-strand scaffold domain of LIGHT or thecysteine-rich repeats domain of HVEM.

The immunogen used to generate the antibody may be any immunogenicportion of LIGHT or HVEM. Preferred immunogens include all or a part ofthe extracellular domain of human LIGHT or HVEM, where these residuescontain the post-translation modifications, such as glycosylation, foundon native LIGHT or HVEM. In other embodiments the immunogen may be theβ-strand scaffold domain of LIGHT or the cysteine-rich repeats domain ofHVEM. Immunogens including the extracellular domain or immunogenicfragments thereof are produced in a variety of ways known in the art,e.g., expression of cloned genes using conventional recombinant methods,synthesized peptide complexes, isolation from cells of origin, cellpopulations expressing high levels of LIGHT or HVEM.

The antibodies disclosed herein are capable of binding to a polypeptidehaving at least about 70%, more preferably 75%, 80%, 85%, 90%, 95%identity to human LIGHT, as found at GENBANK accession numbersNP_(—)003798 (isoform1) and NP_(—)742011 (isoform 2), or HVEM, as foundat GENBANK accession number AAQ89238.

The antibodies may be polyclonal or monoclonal antibodies. Theantibodies may be xenogeneic, allogeneic, syngeneic, or modified formsthereof such as humanized or chimeric antibodies. The antibodies mayalso be antiidiotypic antibodies. Antibodies, as used herein, alsoincludes antibody fragments including Fab and F(ab)₂ fragments, andantibodies produced as a single chain antibody or scFv instead of thenormal multimeric structure. The antibodies may be an IgG such as IgG1,IgG2, IgG3 or IgG4; or IgM, IgA, IgE or IgD isotype. The constant domainof the antibody heavy chain maybe selected depending on the effectorfunction desired. The light chain constant domain may be a kappa orlambda constant domain.

Exemplary antibodies against mouse HVEM are mAbs LBH1 (Xu, et al.,Blood, 109:4097-104 (2007)) and LH1 (Anand, et al. J. Clin. Invest.,116:1045-1051 (2006)). Exemplary antibodies against mouse LIGHT are ML69and ML209 (Tamada, et al. Nat. Med., 6:283-289 (2000)).

Hybridoma cell line LBH1 was deposited by The Johns Hopkins TechnologyTransfer Office on Oct. 18, 2011 under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe purposes of Patent Procedure with the American Type CultureCollection (“ATCC”), Manassas, Va., United States, Patent DepositDesignation PTA-12171.

b. Polypeptides

In another embodiment, LIGHT-HVEM antagonists are polypeptides that bindto LIGHT or HVEM. LIGHT- or HVEM-binding polypeptides can be used toreduce or inhibit the binding of LIGHT to HVEM. LIGHT-HVEM antagonistpolypeptides are also referred to herein as “decoy polypeptides”.Methods of producing polypeptides are well known and within the abilityof one of ordinary skill in the art and are described in more detailbelow.

In some embodiments the polypeptides are soluble fragments of fulllength LIGHT or HVEM polypeptides. As used herein, a fragment of LIGHTor HVEM refers to any subset of the polypeptide that is a shorterpolypeptide of the full length protein. Soluble fragments generally lacksome or all of the intracellular and/or transmembrane domains. In someembodiments, soluble fragments of LIGHT or HVEM include the entireextracellular domains of these proteins. In other embodiments, thesoluble fragments of LIGHT or HVEM include fragments of theextracellular domains of these proteins. In other embodiments, usefulsoluble fragments of LIGHT include the β-strand scaffold domain anduseful soluble fragments of HVEM include the cysteine-rich repeatsdomain.

In another embodiment, the polypeptides are soluble fragments ofreceptors of HVEM other than LIGHT or soluble fragments of receptors ofLIGHT other than HVEM. As discussed above, LIGHT-HVEM antagonistsdisclosed herein do not significantly modulate the binding of LTβ toLTβR. Therefore polypeptide fragments of LTβ or LTβR are specificallyexcluded from the compositions and methods disclosed herein.

Additional receptors for HEVM include lymphotoxin-α (GENBANK accessionnumber AY070490) and hepesvirus gD protein (GENBANK accession numberL09242). An additional receptor for LIGHT is the TR6 receptor (GENBANKaccession number AF134240).

The polypeptide LIGHT-HVEM antagonists can be derived from any speciesof origin. In a preferred embodiment the polypeptide LIGHT-HVEMantagonists are of human origin.

The polypeptides disclosed herein include variant polypeptides. As usedherein, a “variant” polypeptide contains at least one amino acidsequence alteration as compared to the amino acid sequence of thecorresponding wild-type polypeptide. An amino acid sequence alterationcan be, for example, a substitution, a deletion, or an insertion of oneor more amino acids.

A variant LIGHT-HVEM antagonistic polypeptide can have any combinationof amino acid substitutions, deletions or insertions. In one embodiment,isolated LIGHT-HVEM antagonistic variant polypeptides have an integernumber of amino acid alterations such that their amino acid sequenceshares at least 60, 70, 80, 85, 90, 95, 97, 98, 99, 99.5 or 100%identity with an amino acid sequence of a corresponding wild type aminoacid sequence. In a preferred embodiment, LIGHT-HVEM antagonisticvariant polypeptides have an amino acid sequence sharing at least 60,70, 80, 85, 90, 95, 97, 98, 99, 99.5 or 100% identity with the aminoacid sequence of a corresponding wild type polypeptide.

Percent sequence identity can be calculated using computer programs ordirect sequence comparison. Preferred computer program methods todetermine identity between two sequences include, but are not limitedto, the GCG program package, FASTA, BLASTP, and TBLASTN (see, e.g., D.W. Mount, 2001, Bioinformatics: Sequence and Genome Analysis, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The BLASTPand TBLASTN programs are publicly available from NCBI and other sources.The well-known Smith Waterman algorithm may also be used to determineidentity.

Exemplary parameters for amino acid sequence comparison include thefollowing: 1) algorithm from Needleman and Wunsch (J. Mol. Biol.,48:443-453 (1970)); 2) BLOSSUM62 comparison matrix from Hentikoff andHentikoff (Proc. Natl. Acad. Sci. U.S.A., 89:10915-10919 (1992)) 3) gappenalty=12; and 4) gap length penalty=4. A program useful with theseparameters is publicly available as the “gap” program (Genetics ComputerGroup, Madison, Wis.). The aforementioned parameters are the defaultparameters for polypeptide comparisons (with no penalty for end gaps).

Alternatively, polypeptide sequence identity can be calculated using thefollowing equation: % identity=(the number of identicalresidues)/(alignment length in amino acid residues)*100. For thiscalculation, alignment length includes internal gaps but does notinclude terminal gaps.

Amino acid substitutions in LIGHT-HVEM antagonistic variant polypeptidesmay be “conservative” or “non-conservative”. As used herein,“conservative” amino acid substitutions are substitutions wherein thesubstituted amino acid has similar structural or chemical properties,and “non-conservative” amino acid substitutions are those in which thecharge, hydrophobicity, or bulk of the substituted amino acid issignificantly altered. Non-conservative substitutions will differ moresignificantly in their effect on maintaining (a) the structure of thepeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

Examples of conservative amino acid substitutions include those in whichthe substitution is within one of the five following groups: 1) smallaliphatic, nonpolar or slightly polar residues (Ala, Ser, Thr, Pro,Gly); 2) polar, negatively charged residues and their amides (Asp, Asn,Glu, Gln); polar, positively charged residues (His, Arg, Lys); largealiphatic, nonpolar residues (Met, Leu, Ile, Val, Cys); and largearomatic resides (Phe, Tyr, Trp). Examples of non-conservative aminoacid substitutions are those where 1) a hydrophilic residue, e.g., serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.,leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; 2) a cysteine orproline is substituted for (or by) any other residue; 3) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or 4) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) a residue that does not have aside chain, e.g., glycine.

LIGHT-HVEM antagonistic variant polypeptides may be modified by chemicalmoieties that may be present in polypeptides in a normal cellularenvironment, for example, phosphorylation, methylation, amidation,sulfation, acylation, glycosylation, sumoylation and ubiquitylation.LIGHT-HVEM antagonistic variant polypeptides may also be modified with alabel capable of providing a detectable signal, either directly orindirectly, including, but not limited to, radioisotopes and fluorescentcompounds.

LIGHT-HVEM antagonistic variant polypeptides may also be modified bychemical moieties that are not normally added to polypeptides in acellular environment. Such modifications may be introduced into themolecule by reacting targeted amino acid residues of the polypeptidewith an organic derivatizing agent that is capable of reacting withselected side chains or terminal residues. Another modification iscyclization of the protein.

Examples of chemical derivatives of the polypeptides include lysinyl andamino terminal residues derivatized with succinic or other carboxylicacid anhydrides. Derivatization with a cyclic carboxylic anhydride hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate. Carboxyl side groups, aspartyl or glutamyl, may beselectively modified by reaction with carbodiimides (R—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues can be converted to asparaginyl andglutaminyl residues by reaction with ammonia. Polypeptides may alsoinclude one or more D-amino acids that are substituted for one or moreL-amino acids.

The LIGHT-HVEM antagonistic variant polypeptides disclosed herein may becoupled to other polypeptides to form fusion proteins. Provided areLIGHT-HVEM antagonistic variant polypeptides having a first fusionpartner comprising all or a part of a LIGHT-HVEM antagonistic variantpolypeptide fused (i) directly to a second polypeptide or, (ii)optionally, fused to a linker peptide sequence that is fused to thesecond polypeptide. The presence of the fusion partner can alter thesolubility, affinity and/or valency of the LIGHT-HVEM antagonisticvariant polypeptide. As used herein, “valency” refers to the number ofbinding sites available per molecule. LIGHT-HVEM antagonistic variantfusion proteins described herein include any combination of amino acidalteration (i.e. substitution, deletion or insertion), fragment, and/ormodification as described above.

The second polypeptide binding partner may be N-terminal or C-terminalrelative to the LIGHT-HVEM antagonistic variant polypeptide. In apreferred embodiment, the second polypeptide is C-terminal to theLIGHT-HVEM antagonistic variant polypeptide.

A large number of polypeptide sequences that are routinely used asfusion protein binding partners are well known in the art. Examples ofuseful polypeptide binding partners include, but are not limited to,green fluorescent protein (GFP), glutathione S-transferase (GST),polyhistidine, myc, hemagglutinin, Flag™ tag (Kodak, New Haven, Conn.),maltose E binding protein, protein A, and one or more domains of an Igheavy chain constant region, preferably having an amino acid sequencecorresponding to the hinge, C_(H2) and C_(H3) regions of a humanimmunoglobulin Cγ1 chain.

c. Small Molecules and Other Antagonists

It will be appreciated that additional bioactive agents may be screenedfor LIGHT-HVEM antagonistic activity. In one embodiment, candidatebioactive agents are screened for their ability to reduce binding ofLIGHT to HVEM. In another embodiment, candidate bioactive agents arescreened for their ability to reduce activation of either LIGHT or HVEM.The assays preferably utilize human LIGHT and human HVEM proteins,although other LIGHT and HVEM proteins may also be used.

The term “candidate bioactive agent” as used herein describes anymolecule, e.g., protein, small organic molecule, carbohydrates(including polysaccharides), polynucleotide, lipids, etc. Generally aplurality of assay mixtures are run in parallel with different agentconcentrations to obtain a differential response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e., at zero concentration or below the level ofdetection. In addition, positive controls, i.e. the use of agents knownto bind LIGHT or HVEM may be used.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons,more preferably between 100 and 2000, more preferably between about 100and about 1250, more preferably between about 100 and about 1000, morepreferably between about 100 and about 750, more preferably betweenabout 200 and about 500 daltons. Candidate agents comprise functionalgroups necessary for structural interaction with proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups. The candidate agents often comprise cyclical carbon orheterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups. Candidateagents are also found among biomolecules including peptides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof. Particularly preferred arepeptides, e.g., peptidomimetics. Peptidomimetics can be made asdescribed, e.g., in WO 98156401.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs. In a preferred embodiment, the candidate bioactiveagents are organic chemical moieties or small molecule chemicalcompositions, a wide variety of which are available in the art

d. Methods for Measuring LIGHT-HVEM Binding and Activity

Methods for measuring binding affinity of between two molecules, such asLIGHT for HVEM, are well known in the art, and include, but are notlimited to, fluorescence activated cell sorting (FACS), surface plasmonresonance, fluorescence anisotropy, affinity chromatography and affinityselection-mass spectrometry.

Activities of LIGHT or HVEM that can be measured include T cellsurvival, T cell activation, and activation of various transcriptionalfactors including nuclear factor-κB (NF-κB), Jun N-terminal kinase(JNK), and the Jun-containing transcription factor AP-1, throughinteractions with TNF receptor-associated factor (TRAF)-1, 2, 3, 5, and6. T cell activation can be measured as an increase in proliferation orsecretion of cytokines, including, but not limited to, IL-2. Methods formeasuring cell survival, cell proliferation, protein phosphorylationactivation of various transcriptional factors including NF-κB, JNK, andAP-1, and cytokine secretion are well known to those of skill in theart.

2. LIGHT-HVEM Antagonists that Reduce or Inhibit the Expression of LIGHTor HVEM

In another embodiment LIGHT-HVEM antagonists reduce or inhibit theexpression of LIGHT or HVEM. LIGHT-HVEM antagonists that reduce orinhibit expression of LIGHT or HVEM include inhibitory nucleic acids,including, but not limited to, ribozymes, triplex-formingoligonucleotides (TFOs), antisense DNA, siRNA, and microRNA specific fornucleic acids encoding LIGHT or HVEM.

Useful inhibitory nucleic acids include those that reduce the expressionof RNA encoding LIGHT or HVEM by at least 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or 95% compared to controls. Expression of LIGHT or HVEM can bemeasured by methods well know to those of skill in the art, includingnorthern blotting and quantitative polymerase chain reaction (PCR).

Inhibitory nucleic acids and methods of producing them are well known inthe art. siRNA design software is available for example athttp://i.cs.hku.hk/˜sirna/software/sirna.php. Synthesis of nucleic acidsis well known see for example Molecular Cloning: A Laboratory Manual(Sambrook and Russel eds. 3^(rd) ed.) Cold Spring Harbor, N.Y. (2001).The term “siRNA” means a small interfering RNA that is a short-lengthdouble-stranded RNA that is not toxic. Generally, there is no particularlimitation in the length of siRNA as long as it does not show toxicity.“siRNAs” can be, for example, 15 to 49 bp, preferably 15 to 35 bp, andmore preferably 21 to 30 bp long. Alternatively, the double-stranded RNAportion of a final transcription product of siRNA to be expressed canbe, for example, 15 to 49 bp, preferably 15 to 35 bp, and morepreferably 21 to 30 bp long. The double-stranded RNA portions of siRNAsin which two RNA strands pair up are not limited to the completelypaired ones, and may contain nonpairing portions due to mismatch (thecorresponding nucleotides are not complementary), bulge (lacking in thecorresponding complementary nucleotide on one strand), and the like.Nonpairing portions can be contained to the extent that they do notinterfere with siRNA formation. The “bulge” used herein preferablycomprise 1 to 2 nonpairing nucleotides, and the double-stranded RNAregion of siRNAs in which two RNA strands pair up contains preferably 1to 7, more preferably 1 to 5 bulges. In addition, the “mismatch” usedherein is contained in the double-stranded RNA region of siRNAs in whichtwo RNA strands pair up, preferably 1 to 7, more preferably 1 to 5, innumber. In a preferable mismatch, one of the nucleotides is guanine, andthe other is uracil. Such a mismatch is due to a mutation from C to T, Gto A, or mixtures thereof in DNA coding for sense RNA, but notparticularly limited to them. Furthermore, the double-stranded RNAregion of siRNAs in which two RNA strands pair up may contain both bulgeand mismatched, which sum up to, preferably 1 to 7, more preferably 1 to5 in number.

The terminal structure of siRNA may be either blunt or cohesive(overhanging) as long as siRNA can silence, reduce, or inhibit thetarget gene expression due to its RNAi effect. The cohesive(overhanging) end structure is not limited only to the 3′ overhang, andthe 5′ overhanging structure may be included as long as it is capable ofinducing the RNAi effect. In addition, the number of overhangingnucleotide is not limited to the already reported 2 or 3, but can be anynumbers as long as the overhang is capable of inducing the RNAi effect.For example, the overhang consists of 1 to 8, preferably 2 to 4nucleotides. Herein, the total length of siRNA having cohesive endstructure is expressed as the sum of the length of the paireddouble-stranded portion and that of a pair comprising overhangingsingle-strands at both ends. For example, in the case of 19 bpdouble-stranded RNA portion with 4 nucleotide overhangs at both ends,the total length is expressed as 23 bp. Furthermore, since thisoverhanging sequence has low specificity to a target gene, it is notnecessarily complementary (antisense) or identical (sense) to the targetgene sequence. Furthermore, as long as siRNA is able to maintain itsgene silencing effect on the target gene, siRNA may contain a lowmolecular weight RNA (which may be a natural RNA molecule such as tRNA,rRNA or viral RNA, or an artificial RNA molecule), for example, in theoverhanging portion at its one end.

In addition, the terminal structure of the siRNA is not necessarily thecut off structure at both ends as described above, and may have astem-loop structure in which ends of one side of double-stranded RNA areconnected by a linker RNA. The length of the double-stranded RNA region(stem-loop portion) can be, for example, 15 to 49 bp, preferably 15 to35 bp, and more preferably 21 to 30 bp long. Alternatively, the lengthof the double-stranded RNA region that is a final transcription productof siRNAs to be expressed is, for example, 15 to 49 bp, preferably 15 to35 bp, and more preferably 21 to 30 bp long. Furthermore, there is noparticular limitation in the length of the linker as long as it has alength so as not to hinder the pairing of the stem portion. For example,for stable pairing of the stem portion and suppression of therecombination between DNAs coding for the portion, the linker portionmay have a clover-leaf tRNA structure. Even though the linker has alength that hinders pairing of the stem portion, it is possible, forexample, to construct the linker portion to include introns so that theintrons are excised during processing of precursor RNA into mature RNA,thereby allowing pairing of the stem portion. In the case of a stem-loopsiRNA, either end (head or tail) of RNA with no loop structure may havea low molecular weight RNA. As described above, this low molecularweight RNA may be a natural RNA molecule such as tRNA, rRNA or viralRNA, or an artificial RNA molecule.

MiRNAs are produced by the cleavage of short stem-loop precursors byDicer-like enzymes; whereas, siRNAs are produced by the cleavage of longdouble-stranded RNA molecules. MiRNAs are single-stranded, whereassiRNAs are double-stranded.

Methods for producing siRNA are known in the art. Because the sequencefor B7-H4 is known, one of skill in the art could readily produce siRNAsthat downregulate B7-H4 expression in host using the information that ispublicly available.

B. Additional Immunoregulatory Compounds

In certain embodiments, the LIGHT-HVEM antagonists disclosed herein,including antagonistic LIGHT and HVEM antibodies, may be combined withone or more additional therapeutic agents.

The one or more additional therapeutic agents can include agents thatmodulate the activation state of immune system. These agents arereferred to herein as “immunomodulatory” or “immunoregulatory” agents.In one embodiment, the LIGHT-HVEM antagonists described herein can beadministered in combination with immunosuppressive agents, e.g.,antibodies against other lymphocyte surface markers (e.g., CD40) oragainst cytokines, other fusion proteins, e.g., CTLA41g, or otherimmunosuppressive drugs (e.g., cyclosporin A, FK506-like compounds,rapamycin compounds, or steroids).

LIGHT-HVEM antagonists may be combined with immunotherapies based onmodulation of other negative costimulatory pathways, and with CTLA-4modulation in particular. For example, LIGHT-HVEM antagonists may beadvantageously combined with CTLA-4 mimicking agents such as CTLA-41g,which has already found clinical use as an immunosuppressive agent.

As used herein the term “rapamycin compound” includes the neutraltricyclic compound rapamycin, rapamycin derivatives, rapamycin analogs,and other macrolide compounds which are thought to have the samemechanism of action as rapamycin (e.g., inhibition of cytokinefunction). The language “rapamycin compounds” includes compounds withstructural similarity to rapamycin, e.g., compounds with a similarmacrocyclic structure, which have been modified to enhance theirtherapeutic effectiveness. Exemplary Rapamycin compounds are known inthe art (See, e.g. WO 95122972, WO 95116691, WO 95104738, U.S. Pat. Nos.6,015,809; 5,989,591; 5,567,709; 5,559,112; 5,530,006; 5,484,790;5,385,908; 5,202,332; 5,162,333; 5,780,462; 5,120,727).

The language “FK506-like compounds” includes FK506, and FK506derivatives and analogs, e.g., compounds with structural similarity toFK506, e.g., compounds with a similar macrocyclic structure which havebeen modified to enhance their therapeutic effectiveness. Examples ofFK506-like compounds include, for example, those described in WO00101385. Preferably, the language “rapamycin compound” as used hereindoes not include FK506-like compounds.

C. Pharmaceutical Compositions

Pharmaceutical compositions including LIGHT-HVEM antagonists, andvectors containing the same are provided. The pharmaceuticalcompositions may be for administration by oral, parenteral(intramuscular, intraperitoneal, intravenous (IV) or subcutaneousinjection), transdermal (either passively or using iontophoresis orelectroporation), transmucosal (nasal, vaginal, rectal, or sublingual)routes of administration or using bioerodible inserts and can beformulated in dosage forms appropriate for each route of administration.

1. Formulations for Parenteral Administration

In a preferred embodiment, the peptides are administered in an aqueoussolution, by parenteral injection. The formulation may also be in theform of a suspension or emulsion. In general, pharmaceuticalcompositions are provided including effective amounts of a LIGHT-HVEMantagonist, or derivative products, and optionally includepharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions includediluents sterile water, buffered saline of various buffer content (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength; and optionally,additives such as detergents and solubilizing agents (e.g., TWEEN 20,TWEEN 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol). Examples of non-aqueoussolvents or vehicles are propylene glycol, polyethylene glycol,vegetable oils, such as olive oil and corn oil, gelatin, and injectableorganic esters such as ethyl oleate. The formulations may be lyophilizedand redissolved/resuspended immediately before use. The formulation maybe sterilized by, for example, filtration through a bacteria retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions.

2. Formulations for Enteral Administration

LIGHT-HVEM antagonists can be formulated for oral delivery. Oral soliddosage forms are described generally in Remington's PharmaceuticalSciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) atChapter 89. Solid dosage forms include tablets, capsules, pills, trochesor lozenges, cachets, pellets, powders, or granules or incorporation ofthe material into particulate preparations of polymeric compounds suchas polylactic acid, polyglycolic acid, etc. or into liposomes. Suchcompositions may influence the physical state, stability, rate of invivo release, and rate of in vivo clearance of the present proteins andderivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed.(1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which areherein incorporated by reference. The compositions may be prepared inliquid form, or may be in dried powder (e.g., lyophilized) form.Liposomal or proteinoid encapsulation may be used to formulate thecompositions (as, for example, proteinoid microspheres reported in U.S.Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatized with various polymers (e.g., U.S. Pat. No.5,013,556). See also Marshall, K. In: Modern Pharmaceutics Edited by G.S. Banker and C. T. Rhodes Chapter 10, 1979. In general, the formulationwill include the peptide (or chemically modified forms thereof) andinert ingredients which protect peptide in the stomach environment, andrelease of the biologically active material in the intestine.

The polypeptide antagonists may be chemically modified so that oraldelivery of the derivative is efficacious. Generally, the chemicalmodification contemplated is the attachment of at least one moiety tothe component molecule itself, where said moiety permits (a) inhibitionof proteolysis; and (b) uptake into the blood stream from the stomach orintestine. Also desired is the increase in overall stability of thecomponent or components and increase in circulation time in the body.PEGylation is a preferred chemical modification for pharmaceuticalusage. Other moieties that may be used include: propylene glycol,copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone,polyproline, poly-1,3-dioxolane and poly-1,3,6-tioxocane [see, e.g.,Abuchowski and Davis (1981) “Soluble Polymer-Enzyme Adducts,” in Enzymesas Drugs. Hocenberg and Roberts, eds. (Wiley-Interscience: New York,N.Y.) pp. 367-383; and Newmark, et al. (1982) J. Appl. Biochem.4:185-189].

Another embodiment provides liquid dosage forms for oral administration,including pharmaceutically acceptable emulsions, solutions, suspensions,and syrups, which may contain other components including inert diluents;adjuvants such as wetting agents, emulsifying and suspending agents; andsweetening, flavoring, and perfuming agents.

Controlled release oral formulations may be desirable. The LIGHT-HVEMantagonists can be incorporated into an inert matrix which permitsrelease by either diffusion or leaching mechanisms, e.g., gums. Slowlydegenerating matrices may also be incorporated into the formulation.Another form of a controlled release is based on the Oros therapeuticsystem (Alza Corp.), i.e. the drug is enclosed in a semipermeablemembrane which allows water to enter and push drug out through a singlesmall opening due to osmotic effects. For oral formulations, thelocation of release may be the stomach, the small intestine (theduodenum, the jejunem, or the ileum), or the large intestine.Preferably, the release will avoid the deleterious effects of thestomach environment, either by protection of the peptide (or derivative)or by release of the peptide (or derivative) beyond the stomachenvironment, such as in the intestine. To ensure fall gastric resistancea coating impermeable to at least pH 5.0 is essential. Examples of themore common inert ingredients that are used as enteric coatings arecellulose acetate trimellitate (CAT), hydroxypropylmethylcellulosephthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate(PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP),Eudragit L, Eudragit S, and Shellac. These coatings may be used as mixedfilms.

3. Topical Delivery Formulations

Compositions can be applied topically. This does not work well for mostpeptide formulations, although it can be effective especially if appliedto the lungs, nasal, oral (sublingual, buccal), vaginal, or rectalmucosa.

The LIGHT-HVEM antagonists can be delivered to the lungs while inhalingand traverses across the lung epithelial lining to the blood stream whendelivered either as an aerosol or spray dried particles having anaerodynamic diameter of less than about 5 microns.

A wide range of mechanical devices designed for pulmonary delivery oftherapeutic products can be used, including but not limited tonebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art. Some specific examples ofcommercially available devices are the Ultravent nebulizer (MallinckrodtInc., St. Louis, Mo.); the Acorn II nebulizer (Marquest MedicalProducts, Englewood, Colo.); the Ventolin metered dose inhaler (GlaxoInc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler(Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all haveinhalable insulin powder preparations approved or in clinical trialswhere the technology could be applied to the formulations describedherein.

Formulations for administration to the mucosa will typically be spraydried drug particles, which may be incorporated into a tablet, gel,capsule, suspension or emulsion. Standard pharmaceutical excipients areavailable from any formulator. Oral formulations may be in the form ofchewing gum, gel strips, tablets or lozenges.

Transdermal formulations may also be prepared. These will typically beointments, lotions, sprays, or patches, all of which can be preparedusing standard technology. Transdermal formulations will require theinclusion of penetration enhancers.

4. Controlled Delivery Polymeric Matrices

Controlled release polymeric devices can be made for long term releasesystemically following implantation of a polymeric device (rod,cylinder, film, disk) or injection (microparticles). The matrix can bein the form of microparticles such as microspheres, where peptides aredispersed within a solid polymeric matrix or microcapsules, where thecore is of a different material than the polymeric shell, and thepeptide is dispersed or suspended in the core, which may be liquid orsolid in nature. Unless specifically defined herein, microparticles,microspheres, and microcapsules are used interchangeably. Alternatively,the polymer may be cast as a thin slab or film, ranging from nanometersto four centimeters, a powder produced by grinding or other standardtechniques, or even a gel such as a hydrogel.

Either non-biodegradable or biodegradable matrices can be used fordelivery of LIGHT-HVEM antagonists, although biodegradable matrices arepreferred. These may be natural or synthetic polymers, althoughsynthetic polymers are preferred due to the better characterization ofdegradation and release profiles. The polymer is selected based on theperiod over which release is desired. In some cases linear release maybe most useful, although in others a pulse release or “bulk release” mayprovide more effective results. The polymer may be in the form of ahydrogel (typically in absorbing up to about 90% by weight of water),and can optionally be crosslinked with multivalent ions or polymers.

The matrices can be formed by solvent evaporation, spray drying, solventextraction and other methods known to those skilled in the art.Bioerodible microspheres can be prepared using any of the methodsdeveloped for making microspheres for drug delivery, for example, asdescribed by Mathiowitz and Langer, J. Controlled Release 5,13-22(1987); Mathiowitz, et al., Reactive Polymers 6, 275-283 (1987); andMathiowitz, et al., J. Appl. Polymer Sci. 35, 755-774 (1988).

The devices can be formulated for local release to treat the area ofimplantation or injection—which will typically deliver a dosage that ismuch less than the dosage for treatment of an entire body or systemicdelivery. These can be implanted or injected subcutaneously, into themuscle, fat, or swallowed.

III. Methods of Manufacture

A. Methods for Producing Polypeptides

Isolated polypeptides can be obtained by, for example, chemicalsynthesis or by recombinant production in a host cell. To recombinantlyproduce a costimulatory polypeptide, a nucleic acid containing anucleotide sequence encoding the polypeptide can be used to transform,transduce, or transfect a bacterial or eukaryotic host cell (e.g., aninsect, yeast, or mammalian cell). In general, nucleic acid constructsinclude a regulatory sequence operably linked to a nucleotide sequenceencoding a costimulatory polypeptide. Regulatory sequences (alsoreferred to herein as expression control sequences) typically do notencode a gene product, but instead affect the expression of the nucleicacid sequences to which they are operably linked.

Useful prokaryotic and eukaryotic systems for expressing and producingpolypeptides are well know in the art include, for example, Escherichiacoli strains such as BL-21, and cultured mammalian cells such as CHOcells.

In eukaryotic host cells, a number of viral-based expression systems canbe utilized to express polypeptides. Viral based expression systems arewell known in the art and include, but are not limited to, baculoviral,SV40, retroviral, or vaccinia based viral vectors.

Mammalian cell lines that stably express variant costimulatorypolypeptides can be produced using expression vectors with appropriatecontrol elements and a selectable marker. For example, the eukaryoticexpression vectors pCR3.1 (Invitrogen Life Technologies) and p91023(B)(see Wong et al. (1985) Science 228:810-815) are suitable for expressionof variant costimulatory polypeptides in, for example, Chinese hamsterovary (CHO) cells, COS-1 cells, human embryonic kidney 293 cells, NIH3T3cells, BHK21 cells, MDCK cells, and human vascular endothelial cells(HUVEC). Following introduction of an expression vector byelectroporation, lipofection, calcium phosphate, or calcium chlorideco-precipitation, DEAE dextran, or other suitable transfection method,stable cell lines can be selected (e.g., by antibiotic resistance toG418, kanamycin, or hygromycin). The transfected cells can be culturedsuch that the polypeptide of interest is expressed, and the polypeptidecan be recovered from, for example, the cell culture supernatant or fromlysed cells. Alternatively, polypeptides can be produced by (a) ligatingamplified sequences into a mammalian expression vector such as pcDNA3(Invitrogen Life Technologies), and (b) transcribing and translating invitro using wheat germ extract or rabbit reticulocyte lysate.

Polypeptides can be isolated using, for example, chromatographic methodssuch as DEAE ion exchange, gel filtration, and hydroxylapatitechromatography. For example, a polypeptide in a cell culture supernatantor a cytoplasmic extract can be isolated using a protein G column. Insome embodiments, polypeptides can be “engineered” to contain an aminoacid sequence that allows the polypeptides to be captured onto anaffinity matrix. For example, a tag such as c-myc, hemagglutinin,polyhistidine, or Flag™ (Kodak) can be used to aid polypeptidepurification. Such tags can be inserted anywhere within the polypeptide,including at either the carboxyl or amino terminus. Other fusions thatcan be useful include enzymes that aid in the detection of thepolypeptide, such as alkaline phosphatase. Immunoaffinity chromatographyalso can be used to purify polypeptides.

B. Methods for Producing Isolated Nucleic Acid Molecules

Isolated nucleic acid molecules can be produced by standard techniques,including, without limitation, common molecular cloning and chemicalnucleic acid synthesis techniques. For example, polymerase chainreaction (PCR) techniques can be used to obtain an isolated nucleic acidencoding a variant costimulatory polypeptide. PCR is a technique inwhich target nucleic acids are enzymatically amplified. Typically,sequence information from the ends of the region of interest or beyondcan be employed to design oligonucleotide primers that are identical insequence to opposite strands of the template to be amplified. PCR can beused to amplify specific sequences from DNA as well as RNA, includingsequences from total genomic DNA or total cellular RNA. Primerstypically are 14 to 40 nucleotides in length, but can range from 10nucleotides to hundreds of nucleotides in length. General PCR techniquesare described, for example in PCR Primer: A Laboratory Manual, ed. byDieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995.When using RNA as a source of template, reverse transcriptase can beused to synthesize a complementary DNA (cDNA) strand. Ligase chainreaction, strand displacement amplification, self-sustained sequencereplication or nucleic acid sequence-based amplification also can beused to obtain isolated nucleic acids. See, for example, Lewis (1992)Genetic Engineering News 12:1; Guatelli et al. (1990) Proc. Natl. Acad.Sci. USA 87:1874-1878; and Weiss (1991) Science 254:1292-1293.

Isolated nucleic acids can be chemically synthesized, either as a singlenucleic acid molecule or as a series of oligonucleotides (e.g., usingphosphoramidite technology for automated DNA synthesis in the 3′ to 5′direction). For example, one or more pairs of long oligonucleotides(e.g., >100 nucleotides) can be synthesized that contain the desiredsequence, with each pair containing a short segment of complementarity(e.g., about 15 nucleotides) such that a duplex is formed when theoligonucleotide pair is annealed. DNA polymerase can be used to extendthe oligonucleotides, resulting in a single, double-stranded nucleicacid molecule per oligonucleotide pair, which then can be ligated into avector. Isolated nucleic acids can also obtained by mutagenesis. Nucleicacids can be mutated using standard techniques, includingoligonucleotide-directed mutagenesis and/or site-directed mutagenesisthrough PCR. See, Short Protocols in Molecular Biology. Chapter 8, GreenPublishing Associates and John Wiley & Sons, edited by Ausubel et al,1992. Examples of amino acid positions that can be modified includethose described herein.

C. Methods for Producing Antibodies

The basic antibody structural unit comprises a tetramer of subunits.Each tetramer is composed of two identical pairs of polypeptide chains,each pair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta; or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. (See generally,Fundamental Immunology, Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989,Ch. 7).

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame. The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4.

1. Production of Polyclonal Antibodies

Polyclonal antibodies are obtained as sera from immunized animals suchas rabbits, goats, rodents, etc. and may be used directly withoutfurther treatment or may be subjected to conventional enrichment orpurification methods such as ammonium sulfate precipitation, ionexchange chromatography, and affinity chromatography.

2. Production of Monoclonal Antibodies

Monoclonal antibodies may be produced using conventional hybridomatechnology, such as the procedures introduced by Kohler and Milstein(Nature, 256:495-97 (1975)), and modifications thereof. An animal,preferably a mouse, is primed by immunization with an immunogen toelicit the desired antibody response in the primed animal. B lymphocytesfrom the lymph nodes, spleens or peripheral blood of a primed animal arefused with myeloma cells, generally in the presence of a fusionpromoting agent such as polyethylene glycol (PEG). Any of a number ofmurine myeloma cell lines are available for such use: theP3-NS1/1-Ag4-1, P3-x63-k0Ag8.653, Sp2/0-Ag14, or HL1-653 myeloma lines(available from the ATCC, Rockville, Md.). Subsequent steps includegrowth in selective medium so that unfused parental myeloma cells anddonor lymphocyte cells eventually die while only the hybridoma cellssurvive. These are cloned and grown and their supernatants screened forthe presence of antibody of the desired specificity, e.g. by immunoassaytechniques. Positive clones are subcloned, e.g., by limiting dilution,and the monoclonal antibodies are isolated.

Hybridomas produced according to these methods can be propagated invitro or in vivo (in ascites fluid) using techniques known in the art(see generally Fink et al., Prog. Clin. Pathol., 9:121-33 (1984)).Generally, the individual cell line is propagated in culture and theculture medium containing high concentrations of a single monoclonalantibody can be harvested by decantation, filtration, or centrifugation.

a. Production of Chimeric and Humanized Monoclonal Antibodies

Chimeric and humanized antibodies have the same or similar bindingspecificity and affinity as a mouse or other nonhuman antibody thatprovides the starting material for construction of a chimeric orhumanized antibody. Chimeric antibodies are antibodies whose light andheavy chain genes have been constructed, typically by geneticengineering, from immunoglobulin gene segments belonging to differentspecies. For example, the variable (V) segments of the genes from amouse monoclonal antibody may be joined to human constant (C) segments,such as IgG1 and IgG4. Human isotype IgG1 is preferred. In some methods,the isotype of the antibody is human IgG1. IgM antibodies can also beused in some methods. A typical chimeric antibody is thus a hybridprotein consisting of the V or antigen-binding domain from a mouseantibody and the C or effector domain from a human antibody.

Humanized antibodies have variable region framework residuessubstantially from a human antibody (termed an acceptor antibody) andcomplementarity determining regions substantially from a mouse-antibody,(referred to as the donor immunoglobulin). See, Queen et al., Proc.Natl. Acad. Sci. USA 86:10029-10033 (1989), WO 90/07861, U.S. Pat. Nos.5,693,762, 5,693,761, 5,585,089, 5,530,101, and Winter, U.S. Pat. No.5,225,539). The constant region(s), if present, are also substantiallyor entirely from a human immunoglobulin. The human variable domains areusually chosen from human antibodies whose framework sequences exhibit ahigh degree of sequence identity with the murine variable region domainsfrom which the CDRs were derived. The heavy and light chain variableregion framework residues can be derived from the same or differenthuman antibody sequences. The human antibody sequences can be thesequences of naturally occurring human antibodies or can be consensussequences of several human antibodies. Certain amino acids from thehuman variable region framework residues are selected for substitutionbased on their possible influence on CDR conformation and/or binding toantigen. Investigation of such possible influences is by modeling,examination of the characteristics of the amino acids at particularlocations, or empirical observation of the effects of substitution ormutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid should usually be substituted bythe equivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

(1) noncovalently binds antigen directly,

(2) is adjacent to a CDR region,

(3) otherwise interacts with a CDR region (e.g. is within about 6 A of aCDR region), or

(4) participates in the VL-VH interface.

Other candidates for substitution are acceptor human framework aminoacids that are unusual for a human immunoglobulin at that position.These amino acids can be substituted with amino acids from theequivalent position of the mouse donor antibody or from the equivalentpositions of more typical human immunoglobulins. Other candidates forsubstitution are acceptor human framework amino acids that are unusualfor a human immunoglobulin at that position. The variable regionframeworks of humanized immunoglobulins usually show at least 85%sequence identity to a human variable region framework sequence orconsensus of such sequences.

b. Production of Human Monoclonal Antibodies

Human antibodies against LIGHT and HVEM can be produced by a variety oftechniques described below. Some human antibodies are selected bycompetitive binding experiments or otherwise, to have the same epitopespecificity as a particular mouse antibody. Human antibodies preferablyhave isotype specificity human IgG1.

One method for producing human monoclonal antibodies is the triomamethodology. The basic approach and an exemplary cell fusion partner,SPAZ-4, for use in this approach have been described by Oestberg et al.,Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; andEngleman et al., U.S. Pat. No. 4,634,666). The antibody-producing celllines obtained by this method are called triomas, because they aredescended from three cells—two human and one mouse. Initially, a mousemyeloma line is fused with a human B-lymphocyte to obtain anon-antibody-producing xenogeneic hybrid cell, such as the SPAZ-4 cellline. The xenogeneic cell is then fused with an immunized humanB-lymphocyte to obtain an antibody-producing trioma cell line. Triomashave been found to produce antibody more stably than ordinary hybridomasmade from human cells.

The immunized B-lymphocytes are obtained from the blood, spleen, lymphnodes or bone marrow of a human donor. If antibodies against a specificantigen or epitope are desired, it is preferable to use that antigen orepitope thereof for immunization. Immunization can be either in vivo orin vitro. For in vivo immunization, B cells are typically isolated froma human immunized with LIGHT or HVEM. In some methods, B cells areisolated from the same patient who is ultimately to be administeredantibody therapy. For in vitro immunization, B-lymphocytes are typicallyexposed to antigen for a period of 7-14 days in a media such asRPMI-1640 supplemented with 10% human plasma.

The immunized B-lymphocytes are fused to a xenogeneic hybrid cell suchas SPAZ-4 by well known methods. For example, the cells are treated with40-50% polyethylene glycol of MW 1000-4000, at about 37°0 C., for about5-10 min. Cells are separated from the fusion mixture and propagated inmedia selective for the desired hybrids (e.g., HAT or AH). Clonessecreting antibodies having the required binding specificity areidentified by assaying the trioma culture medium for the ability to bindto LIGHT or HVEM. Triomas producing human antibodies having the desiredspecificity are subcloned by the limiting dilution technique and grownin vitro in culture medium. The trioma cell lines obtained are thentested for the ability to bind LIGHT or HVEM.

Although triomas are genetically stable they do not produce antibodiesat very high levels. Expression levels can be increased by cloningantibody genes from the trioma into one or more expression vectors, andtransforming the vector into standard mammalian, bacterial or yeast celllines.

Human antibodies against LIGHT and HVEM can also be produced fromnon-human transgenic mammals having transgenes encoding at least asegment of the human immunoglobulin locus. Usually, the endogenousimmunoglobulin locus of such transgenic mammals is functionallyinactivated. Preferably, the segment of the human immunoglobulin locusincludes unrearranged sequences of heavy and light chain components.Both inactivation of endogenous immunoglobulin genes and introduction ofexogenous immunoglobulin genes can be achieved by targeted homologousrecombination, or by introduction of YAC chromosomes. The transgenicmammals resulting from this process are capable of functionallyrearranging the immunoglobulin component sequences, and expressing arepertoire of antibodies of various isotypes encoded by humanimmunoglobulin genes, without expressing endogenous immunoglobulingenes. The production and properties of mammals having these propertiesare described in detail by, e.g., Lonberg et al., WO93/1222, U.S. Pat.Nos. 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429, 5,661,016,5,633,425, 5,625,126, 5,569,825, 5,545,806, Nature 148, 1547-1553(1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741.Transgenic mice are particularly suitable. Anti-LIGHT and anti-HVEMantibodies are obtained by immunizing a transgenic nonhuman mammal withpolypeptides corresponding to fill length LIGHT or HVEM polypeptides orimmunogenic fragments thereof. Monoclonal antibodies are prepared by,e.g., fusing B-cells from such mammals to suitable myeloma cell linesusing conventional Kohler-Milstein technology. Human polyclonalantibodies can also be provided in the form of serum from humansimmunized with an immunogenic agent. Optionally, such polyclonalantibodies can be concentrated by affinity purification using LIGHT orHVEM polypeptides or fragments thereof as an affinity reagent.

A further approach for obtaining human anti-LIGHT and anti-HVEMantibodies is to screen a DNA library from human B cells according tothe general protocol outlined by Huse et al., Science 246:1275-1281(1989). As described for trioma methodology, such B cells can beobtained from a human immunized with full length LIGHT or HVEMpolypeptides or immunogenic fragments thereof. Optionally, such B cellsare obtained from a patient who is ultimately to receive antibodytreatment. Antibodies binding to LIGHT, HVEM, or fragments thereof areselected. Sequences encoding such antibodies (or binding fragments) arethen cloned and amplified. The protocol described by Huse is renderedmore efficient in combination with phage-display technology. See, e.g.,Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, U.S. Pat.Nos. 5,877,218, 5,871,907, 5,858,657, 5,837,242, 5,733,743 and5,565,332). In these methods, libraries of phage are produced in whichmembers display different antibodies on their outer surfaces. Antibodiesare usually displayed as Fv or Fab fragments. Phage displayingantibodies with a desired specificity are selected by affinityenrichment to a LIGHT or HVEM polypeptide or fragment thereof.

In a variation of the phage-display method, human antibodies having thebinding specificity of a selected murine antibody can be produced(Winter, WO 92/20791). In this method, either the heavy or light chainvariable region of the selected murine antibody is used as a startingmaterial. If, for example, a light chain variable region is selected asthe starting material, a phage library is constructed in which membersdisplay the same light chain variable region (i.e., the murine startingmaterial) and a different heavy chain variable region. The heavy chainvariable regions are obtained from a library of rearranged human heavychain variable regions. A phage showing strong specific binding for αSyn(e.g., at least 10⁸ and preferably at least 10⁹M⁻¹) is selected. Thehuman heavy chain variable region from this phage then serves as astarting material for constructing a further phage library. In thislibrary, each phage displays the same heavy chain variable region (i.e.,the region identified from the first display library) and a differentlight chain variable region. The light chain variable regions areobtained from a library of rearranged human variable light chainregions. Again, phage showing strong specific binding for LIGHT or HVEMare selected. These phage display the variable regions of completelyhuman anti-LIGHT or anti-HVEM antibodies. These antibodies usually havethe same or similar epitope specificity as the murine starting material.

The heavy and light chain variable regions of chimeric, humanized, orhuman antibodies can be linked to at least a portion of a human constantregion. The choice of constant region depends, in part, whetherantibody-dependent complement and/or cellular mediated toxicity isdesired. For example, isotopes IgG1 and IgG3 have complement activityand isotypes IgG2 and IgG4 do not. Choice of isotype can also affectpassage of antibody into the brain. Human isotype IgG1 is preferred.Light chain constant regions can be lambda or kappa. Antibodies can beexpressed as tetramers containing two light and two heavy chains, asseparate heavy chains, light chains, as Fab, Fab′ F(ab′)2, and Fv, or assingle chain antibodies in which heavy and light chain variable domainsare linked through a spacer.

3. Expression of Recombinant Antibodies

Chimeric, humanized and human antibodies are typically produced byrecombinant expression. Recombinant polynucleotide constructs typicallyinclude an expression control sequence operably linked to the codingsequences of antibody chains, including naturally associated orheterologous promoter regions. Preferably, the expression controlsequences are eukaryotic promoter systems in vectors capable oftransforming or transfecting eukaryotic host cells. Once the vector hasbeen incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the crossreactingantibodies. These expression vectors are typically replicable in thehost organisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors contain selection markers,e.g., ampicillin-resistance or hygromycin-resistance, to permitdetection of those cells transformed with the desired DNA sequences.

E. coli is one prokaryotic host particularly useful for cloning the DNAsequences of the present invention. Microbes, such as yeast are alsouseful for expression. Saccharomyces is a preferred yeast host, withsuitable vectors having expression control sequences, an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

Mammalian cells are a preferred host for expressing nucleotide segmentsencoding immunoglobulins or fragments thereof (Winnacker, From Genes toClones, VCH Publishers, NY, 1987). A number of suitable host cell linescapable of secreting intact heterologous proteins have been developed inthe art, and include CHO cell lines, various COS cell lines, HeLa cells,L cells, human embryonic kidney cell, and myeloma cell lines.Preferably, the cells are nonhuman. Expression vectors for these cellscan include expression control sequences, such as an origin ofreplication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49(1986)), and necessary processing information sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from endogenous genes includingcytomegalovirus, SV40, adenovirus, bovine papillomavirus (Co et al., J.Immunol. 148:1149 (1992).

Alternatively, antibody coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,U.S. Pat. Nos. 5,741,957, 5,304,489, 5,849,992). Suitable transgenesinclude coding sequences for light and/or heavy chains in operablelinkage with a promoter and enhancer from a mammary gland specific gene,such as casein or beta lactoglobulin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by well-known methods, depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment,electroporation, lipofection, biolistics or viral-based transfection canbe used for other cellular hosts. Other methods used to transformmammalian cells include the use of polybrene, protoplast fusion,liposomes, electroporation, and microinjection (see generally, Sambrooket al., supra). For production of transgenic animals, transgenes can bemicroinjected into fertilized oocytes, or can be incorporated into thegenome of embryonic stem cells, and the nuclei of such cells transferredinto enucleated oocytes.

Once expressed, antibodies can be purified according to standardprocedures of the art, including HPLC purification, columnchromatography, gel electrophoresis and the like (see generally, Scopes,Protein Purification (Springer-Verlag, NY, 1982)).

Polypeptide immunogens disclosed herein can also be linked to a suitablecarrier molecule to form a conjugate which helps elicit an immuneresponse. Suitable carriers include serum albumins, keyhole limpethemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanustoxoid, or a toxoid from other pathogenic bacteria, such as diphtheria,E. coli, cholera, or H. pylori, or an attenuated toxin derivative. Tcell epitopes are also suitable carrier molecules. Some conjugates canbe formed by linking agents of the invention to an immunostimulatorypolymer molecule (e.g., tripalmitoyl-S-glycerine cysteine(Pam.sub.3Cys), mannan (a manose polymer), or glucan (a beta 1.fwdarw.2polymer)), cytokines (e.g., IL-1, IL-1 alpha and beta peptides, IL-2,gamma-INF, IL-10, GM-CSF), and chemokines (e.g., MIP1alpha and beta, andRANTES). Immunogenic agents can also be linked to peptides that enhancetransport across tissues, as described in O'Mahony, WO 97/17613 and WO97/17614. Immunogens may be linked to the carries with or with outspacers amino acids (e.g., gly-gly).

Some conjugates can be formed by linking agents to at least one T cellepitope. Some T cell epitopes are promiscuous while other T cellepitopes are universal. Promiscuous T cell epitopes are capable ofenhancing the induction of T cell immunity in a wide variety of subjectsdisplaying various HLA types. In contrast to promiscuous T cellepitopes, universal T cell epitopes are capable of enhancing theinduction of T cell immunity in a large percentage, e.g., at least 75%,of subjects displaying various HLA molecules encoded by different HLA-DRalleles.

A large number of naturally occurring T-cell epitopes exist, such as,tetanus toxoid (e.g., the P2 and P30 epitopes), Hepatitis B surfaceantigen, pertussis, toxoid, measles virus F protein, Chlamydiatrachomitis major outer membrane protein, diphtheria toxoid, Plasmodiumfalciparum circumsporozite T, Plasmodium falciparum CS antigen,Schistosoma mansoni triose phosphate isomersae, Escherichia coli TraT,and Influenza virus hemagluttinin (HA). The immunogenic peptides of theinvention can also be conjugated to the T-cell epitopes described inSinigaglia F. et al., Nature, 336:778-780 (1988); Chicz R. M. et al., J.Exp. Med., 178:27-47 (1993); Hammer J. et al., Cell 74:197-203 (1993);Falk K. et al., Immunogenetics, 39:230-242 (1994); WO 98/23635; and,Southwood S. et al. J. Immunology, 160:3363-3373 (1998).

Alternatively, the conjugates can be formed by linking agents to atleast one artificial T-cell epitope capable of binding a largeproportion of MHC Class II molecules., such as the pan DR epitope(“PADRE”). PADRE is described in U.S. Pat. No. 5,736,142, WO 95/07707,and Alexander J et al., Immunity, 1:751-761 (1994). A preferred PADREpeptide is AKXVAAWTLKAAA, wherein X is preferably cyclohexylalanine,tyrosine or phenylalanine, with cyclohexylalanine being most preferred.

Immunogenic agents can be linked to carriers by chemical crosslinking.Techniques for linking an immunogen to a carrier include the formationof disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio)propionate(SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC) (if the peptide lacks a sulfhydryl group, this can be provided byaddition of a cysteine residue). These reagents create a disulfidelinkage between themselves and peptide cysteine resides on one proteinand an amide linkage through the epsilon-amino on a lysine, or otherfree amino group in other amino acids. A variety of suchdisulfide/amide-forming agents are described by Immun. Rev. 62, 185(1982). Other bifunctional coupling agents form a thioether rather thana disulfide linkage. Many of these thio-ether-forming agents arecommercially available and include reactive esters of 6-maleimidocaproicacid, 2-bromoacetic acid, and 2-iodoacetic acid,4-(N-maleimido-methyl)cy-clohexane-1-carboxylic acid. The carboxylgroups can be activated by combining them with succinimide or1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.

Immunogenicity can be improved through the addition of spacer residues(e.g., Gly-Gly) between the T_(h) epitope and the peptide immunogen. Inaddition to physically separating the T_(h) epitope from the B cellepitope (i.e., the peptide immunogen), the glycine residues can disruptany artificial secondary structures created by the joining of the T_(h)epitope with the peptide immunogen, and thereby eliminate interferencebetween the T and/or B cell responses. The conformational separationbetween the helper epitope and the antibody eliciting domain thuspermits more efficient interactions between the presented immunogen andthe appropriate T_(h) and B cells.

To enhance the induction of T cell immunity in a large percentage ofsubjects displaying various HLA types to an agent of the presentinvention, a mixture of conjugates with different T_(h) cell epitopescan be prepared. The mixture may contain a mixture of at least twoconjugates with different T_(h) cell epitopes, a mixture of at leastthree conjugates with different T_(h) cell epitopes, or a mixture of atleast four conjugates with different T_(h) cell epitopes. The mixturemay be administered with an adjuvant.

Immunogenic peptides can also be expressed as fusion proteins withcarriers (i.e., heterologous peptides). The immunogenic peptide can belinked at its amino terminus, its carboxyl terminus, or both to acarrier. Optionally, multiple repeats of the immunogenic peptide can bepresent in the fusion protein. Optionally, an immunogenic peptide can belinked to multiple copies of a heterologous peptide, for example, atboth the N and C termini of the peptide. Some carrier peptides serve toinduce a helper T-cell response against the carrier peptide. The inducedhelper T-cells in turn induce a B-cell response against the immunogenicpeptide linked to the carrier peptide.

IV. Methods of Use

A. Reducing or Inhibiting T Cell Activation or Survival

LIGHT-HVEM antagonists, nucleic acids encoding LIGHT-HVEM antagonists,or cells expressing LIGHT-HVEM antagonists can be used to reduce orinhibit the activation of T cells (i.e., decrease antigen-specificproliferation of T cells, decrease cytokine production by T cells,inhibit or reduce differentiation and effector functions of T cells) orto reduce T cell survival.

LIGHT-HVEM antagonists can be used to reduce or inhibit T cellactivation and/or survival. The methods can include contacting a T cellwith a LIGHT-HVEM antagonists. The contacting can be in vitro ex vivo,or in vivo (e.g., in a mammal such as a mouse, rat, rabbit, dog, cow,pig, non-human primate, or a human).

The contacting can occur before, during, or after activation of the Tcell. Typically, contacting of the T cell with LIGHT-HVEM antagonistscan after T cell activation. Activation can be, for example, by exposingthe T cell to an antibody that binds to the T cell receptor (TCR) or oneof the polypeptides of the CD3 complex that is physically associatedwith the TCR. Alternatively, a T cell can be exposed to either analloantigen (e.g., a MHC alloantigen) on, for example, an APC [e.g., aninterdigitating dendritic cell (referred to herein as a dendritic cell),a macrophage, a monocyte, or a B cell] or an antigenic peptide producedby processing of a protein antigen by any of the above APC and presentedto the T cell by MHC molecules on the surface of the APC. The T cell canbe a CD4⁺ T cell or a CD8⁺ T cell.

The LIGHT-HVEM antagonists can be any of those described herein,including any of the disclosed amino acid alterations, polypeptidefragments, fusion proteins and combinations thereof.

In vitro application of the LIGHT-HVEM antagonists can be useful, forexample, in basic scientific studies of immune mechanisms.

B. Therapeutic Uses of LIGHT-HVEM Antagonists

1. Conditions to be Treated

The LIGHT-HVEM antagonists provided herein are generally useful asimmune response-reducing therapeutics. In general, the compositions areuseful for treating a subject having or being predisposed to any diseaseor disorder to which the subject's immune system mounts an immuneresponse. The ability of LIGHT-HVEM antagonists to reduce the activationand/or survival of T cells makes the disclosed compositions useful toreduce or inhibit immune responses involving T cells. The terms “treat”and “treating”, as used herein includes alleviating, preventing and/oreliminating one or more symptoms associated with inflammatory immuneresponses, autoimmune disorders or immune responses to grafts, includinggraft-versus-host disease.

In one embodiment, the compositions and methods disclosed herein areuseful for the treatment or prevention of graft rejection or graftversus host disease. The methods and compositions disclosed herein canbe used in the prevention or treatment of any type of allograftrejection or graft versus host disease for any type of graft, includinga xenograft. The allograft can be an organ transplant, such as, but notlimited to, a heart, kidney, liver, lung or pancreas. Alternatively, theallograft can be a tissue transplant, such as, but not limited to, heartvalve, endothelial, cornea, eye lens or bone marrow tissue transplant.In yet other embodiments, the allograft can be a skin graft.

In another embodiment, the compositions and methods disclosed herein areuseful for the treatment or prevention of inflammatory immune responsesand autoimmune disorders. Representative inflammatory and autoimmunedisorders that can be treated include, but are not limited to,rheumatoid arthritis, systemic lupus erythematosus, alopecia areata,anklosing spondylitis, antiphospholipid syndrome, autoimmune addison'sdisease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmuneinner ear disease, autoimmune lymphoproliferative syndrome (alps),autoimmune thrombocytopenic purpura (ATP), Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatiguesyndrome immune deficiency, syndrome (CFIDS), chronic inflammatorydemyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinindisease, Crest syndrome, Crohn's disease, Dego's disease,dermatomyositis, dermatomyositis—juvenile, discoid lupus, essentialmixed cryoglobulinemia, fibromyalgia—fibromyositis, grave's disease,guillain-barre, hashimoto's thyroiditis, idiopathic pulmonary fibrosis,idiopathic thrombocytopenia purpura (ITP), Iga nephropathy, insulindependent diabetes (Type I), inflammatory bowel disease (IBD), juvenilearthritis, Meniere's disease, mixed connective tissue disease, multiplesclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia,polyarteritis nodosa, polychondritis, polyglancular syndromes,polymyalgia rheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud'sphenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis,scleroderma, Sjogren's syndrome, stiff-man syndrome, Takayasu arteritis,temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis,vasculitis, vitiligo, and Wegener's granulomatosis.

B. Methods of Treating or Preventing Immunologic Disorders

It has been discovered that LIGHT and HVEM promote T cell activation andsurvival during immunologic responses. It is believed that LIGHT-HVEMbinding of T cells is the mechanism for promoting activation andsurvival. LIGHT-HVEM antagonists include compounds that inhibit, reduce,or block the biological activity or expression of LIGHT and/or HVEM.

Therefore, one embodiment of a method for treating or inhibitingimmunologic disorders, including inflammatory disorders, autoimmunedisorders, and immune responses involved in graft rejection, includinggraft-versus-host disease, is by interfering with the binding of LIGHTto HVEM and by antagonizing their activity. For example, the method canbe by administering to a host in need thereof an effective amount of oneor more LIGHT-HVEM antagonists. In one embodiment, interference withLIGHT-HVEM binding and associated biological activities is accomplishedby providing one or more LIGHT-HVEM antagonists that reduce or inhibitbinding of LIGHT to HVEM. In another embodiment, LIGHT and/or HVEMexpression is downregulated by providing one or more inhibitory nucleicacids including, but not limited to, ribozymes, triplex-formingoligonucleotides (TFOs), antisense DNA, siRNA, and microRNA specific fornucleic acids encoding LIGHT or HVEM. LIGHT-HVEM antagonists can also beprovided in combination with other immunomodulatory agents, such asthose described above.

The compositions and methods disclosed herein can be used forprophylactic and therapeutic applications. In prophylactic applications,LIGHT-HVEM antagonists are provided in amounts and frequencies ofadministration sufficient to eliminate or reduce the risk or delay theoutset of immunologic disorders, including physiological, biochemical,histologic and/or behavioral symptoms of the disorder, its complicationsand intermediate pathological phenotypes presenting during developmentof the disease or disorder. In therapeutic applications, thecompositions and methods disclosed herein are administered to a patientsuspected of or already suffering from such an immunologic disease ordisorder to treat, at least partially, the symptoms of the disease(physiological, biochemical, histologic and/or behavioral), includingits complications and intermediate pathological phenotypes indevelopment of the disease or disorder. An amount adequate to accomplishtherapeutic or prophylactic treatment is defined as a therapeutically-or prophylactically-effective amount.

With respect to allograft rejection or graft versus host disease, in apreferred embodiment, the prophylactic methods are initiated prior totransplantation of the allograft. In certain embodiments, the methodscan be practiced for a day, three days, a week, two weeks or a monthprior to a transplantation. In other embodiments, the drugs areadministered for a week, two weeks, three weeks, one month, two months,three months or six months following a transplantation. In a preferredembodiment, the methods are practiced both before and after atransplantation is carried out.

The outcome of the therapeutic and prophylactic methods disclosed hereinis to at least produce in a patient a healthful benefit, which includes,but is not limited to, prolonging the lifespan of a patient, prolongingthe onset of symptoms of the disorder, and/or alleviating a symptom ofthe disorder after onset of a symptom of the disorder. For example, inthe context of allograft rejection, the therapeutic and prophylacticmethods can result in prolonging the lifespan of an allograft recipient,prolonging the duration of allograft tolerance prior to rejection,and/or alleviating a symptom associated with allograft rejection.

C. Methods of Administration of LIGHT-HVEM Antagonists

In some in vivo approaches, a LIGHT-HVEM antagonist itself isadministered to a subject in a therapeutically effective amount.Typically, the polypeptides can be suspended in apharmaceutically-acceptable carrier. Pharmaceutically acceptablecarriers are biologically compatible vehicles (e.g., physiologicalsaline) that are suitable for administration to a human. Atherapeutically effective amount is an amount of a LIGHT-HVEM antagonistthat is capable of producing a medically desirable result (e.g., reducedT cell activation of survival) in a treated animal. LIGHT-HVEMantagonists can be administered orally or by intravenous infusion, orinjected subcutaneously, intramuscularly, intraperitoneally,intrarectally, intravaginally, intranasally, intragastrically,intratracheally, or intrapulmonarily. The LIGHT-HVEM antagonists can bedelivered directly to an appropriate lymphoid tissue (e.g., spleen,lymph node, or mucosal-associated lymphoid tissue) or directly to anorgan or tissue graft.

D. Methods of Administration of Nucleic Acids and Cells

Nucleic acids encoding LIGHT-HVEM antagonists can be administered tosubjects in need thereof. Nucleic delivery involves introduction of“foreign” nucleic acids into a cell and ultimately, into a live animal.Several general strategies for gene therapy have been studied and havebeen reviewed extensively (Yang, N-S., Crit. Rev. Biotechnol. 12:335-356(1992); Anderson, W. F., Science 256:808-813 (1992); Miller, A. S.,Nature 357:455-460 (1992); Crystal, R. G., Amer. J. Med. 92(suppl6A):44S-52S (1992); Zwiebel, J. A. et al., Ann. N.Y. Acad. Sci.618:394-404 (1991); McLachlin, J. R. et al., Prog. Nuc. Acid Res. Molec.Biol. 38:91-135 (1990); Kohn, D. B. et al., Cancer Invest. 7:179-192(1989), which references are herein incorporated by reference in theirentirety).

One approach includes nucleic acid transfer into primary cells inculture followed by autologous transplantation of the ex vivotransformed cells into the host, either systemically or into aparticular organ or tissue. In one embodiment, vectors containingnucleic acids encoding LIGHT-HVEM antagonists are transfected into cellsthat are administered to a subject in need thereof.

Ex vivo methods can include, for example, the steps of harvesting cellsfrom a subject, culturing the cells, transducing them with an expressionvector, and maintaining the cells under conditions suitable forexpression of the variant costimulatory polypeptides provided herein.These methods are known in the art of molecular biology. Thetransduction step can be accomplished by any standard means used for exvivo gene therapy, including, for example, calcium phosphate,lipofection, electroporation, viral infection, and biolistic genetransfer. Alternatively, liposomes or polymeric microparticles can beused. Cells that have been successfully transduced then can be selected,for example, for expression of the coding sequence or of a drugresistance gene. The cells then can be lethally irradiated (if desired)and injected or implanted into the subject.

Nucleic acid therapy can be accomplished by direct transfer of afunctionally active DNA into mammalian somatic tissue or organ in vivo.For example, nucleic acids encoding LIGHT-HVEM antagonists can beadministered directly to lymphoid tissues. Alternatively, lymphoidtissue specific targeting can be achieved using lymphoid tissue-specifictranscriptional regulatory elements (TREs) such as a B lymphocyte-, Tlymphocyte-, or dendritic cell-specific TRE. Lymphoid tissue specificTREs include, for example, those known in the art [see, e.g., Thompsonet al. (1992) Mol. Cell. Biol. 12:1043-1053; Todd et al. (1993) J. Exp.Med. 177:1663-1674; and Penix et al. (1993) J. Exp. Med. 178:1483-1496].

DNA transfer can be achieved using a number of approaches describedbelow. These systems can be tested for successful expression in vitro byuse of a selectable marker (e.g., G418 resistance) to select transfectedclones expressing the DNA, followed by detection of the presence of theLIGHT-HVEM antagonist expression product (after treatment with theinducer in the case of an inducible system) using an antibody to theproduct in an appropriate immunoassay. Efficiency of the procedure,including DNA uptake, plasmid integration and stability of integratedplasmids, can be improved by linearizing the plasmid DNA using knownmethods, and co-transfection using high molecular weight mammalian DNAas a “carrier”.

Examples of successful “gene transfer” reported in the art include: (a)direct injection of plasmid DNA into mouse muscle tissues, which led toexpression of marker genes for an indefinite period of time (Wolff, J.A. et al., Science 247:1465 (1990); Acsadi, G. et al., The New Biologist3:71 (1991)); (b) retroviral vectors are effective for in vivo and insitu infection of blood vessel tissues; (c) portal vein injection anddirect injection of retrovirus preparations into liver effected genetransfer and expression in vivo (Horzaglou, M. et al., J. Biol. Chem.265:17285 (1990); Koleko, M. et al., Human Gene Therapy 2:27 (1991);Ferry, N. et al., Proc. Natl. Acad. Sci. USA 88:8387 (1991)); (d)intratracheal infusion of recombinant adenovirus into lung tissues waseffective for in vivo transfer and prolonged expression of foreign genesin lung respiratory epithelium (Rosenfeld, M. A. et al., Science 252:431(1991); (e) Herpes simplex virus vectors achieved in vivo gene transferinto brain tissue (Ahmad, F. et al., eds, Miami Short Reports—Advancesin Gene Technology: The Molecular Biology of Human Genetic Disease, Vol1, Boerringer Manneheim Biochemicals, USA, 1991).

Retroviral-mediated human therapy utilizes amphotrophic,replication-deficient retrovirus systems (Temin, H. M., Human GeneTherapy 1:111 (1990); Temin et al., U.S. Pat. No. 4,980,289; Temin etal., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No. No. 5,124,263;Wills, J. W. U.S. Pat. No. 5,175,099; Miller, A. D., U.S. Pat. No.4,861,719). Such vectors have been used to introduce functional DNA intohuman cells or tissues, for example, the adenosine deaminase gene intolymphocytes, the NPT-II gene and the gene for tumor necrosis factor intotumor infiltrating lymphocytes. Retrovirus-mediated gene deliverygenerally requires target cell proliferation for gene transfer (Miller,D. G. et al., Mol. Cell. Biol. 10:4239 (1990). This condition is met bycertain of the preferred target cells into which the present DNAmolecules are to be introduced, i.e., actively growing tumor cells. Genetherapy of cystic fibrosis using transfection by plasmids using any of anumber of methods and by retroviral vectors has been described byCollins et al., U.S. Pat. No. 5,240,846.

Nucleic acid molecules encoding LIGHT-HVEM antagonists may be packagedinto retrovirus vectors using packaging cell lines that producereplication-defective retroviruses, as is well-known in the art (see,for example, Cone, R. D. et al., Proc. Natl. Acad. Sci. USA 81:6349-6353(1984); Mann, R. F. et al., Cell 33:153-159 (1983); Miller, A. D. etal., Molec. Cell. Biol. 5:431-437 (1985),; Sorge, J., et al., Molec.Cell. Biol. 4:1730-1737 (1984); Hock, R. A. et al., Nature 320:257(1986); Miller, A. D. et al., Molec. Cell. Biol. 6:2895-2902 (1986).Newer packaging cell lines which are efficient and safe for genetransfer have also been described (Bank et al., U.S. Pat. No.5,278,056).

This approach can be utilized in a site specific manner to deliver theretroviral vector to the tissue or organ of choice. Thus, for example, acatheter delivery system can be used (Nabel, E. G et al., Science244:1342 (1989)). Such methods, using either a retroviral vector or aliposome vector, are particularly useful to deliver the nucleic acid tobe expressed to a blood vessel wall, or into the blood circulation of atumor.

Other virus vectors may also be used, including recombinant adenoviruses(Horowitz, M. S., In: Virology, Fields, B N et al., eds, Raven Press,New York, 1990, p. 1679; Berkner, K. L., Biotechniques 6:616 9191988),Strauss, S. E., In: The Adenoviruses, Ginsberg, H S, ed., Plenum Press,New York, 1984, chapter 11), herpes simplex virus (HSV) forneuron-specific delivery and persistence. Advantages of adenovirusvectors for human gene therapy include the fact that recombination israre, no human malignancies are known to be associated with suchviruses, the adenovirus genome is double stranded DNA which can bemanipulated to accept foreign genes of up to 7.5 kb in size, and liveadenovirus is a safe human vaccine organisms. Adeno-associated virus isalso useful for human therapy (Samulski, R. J. et al., EMBO J. 10:3941(1991).

Another vector which can express the disclosed DNA molecule and isuseful in the present therapeutic setting, particularly in humans, isvaccinia virus, which can be rendered non-replicating (U.S. Pat. Nos.5,225,336; 5,204,243; 5,155,020; 4,769,330; Sutter, G et al., Proc.Natl. Acad. Sci. USA (1992) 89:10847-10851; Fuerst, T. R. et al., Proc.Natl. Acad. Sci. USA (1989) 86:2549-2553; Falkner F. G. et al.; Nucl.Acids Res (1987) 15:7192; Chakrabarti, S et al., Molec. Cell. Biol.(1985) 5:3403-3409). Descriptions of recombinant vaccinia viruses andother viruses containing heterologous DNA and their uses in immunizationand DNA therapy are reviewed in: Moss, B., Curr. Opin. Genet. Dev.(1993) 3:86-90; Moss, B. Biotechnology (1992) 20: 345-362; Moss, B.,Curr Top Microbiol Immunol (1992) 158:25-38; Moss, B., Science (1991)252:1662-1667; Piccini, A et al., Adv. Virus Res. (1988) 34:43-64; Moss,B. et al., Gene Amplif Anal (1983) 3:201-213.

In addition to naked DNA or RNA, or viral vectors, engineered bacteriamay be used as vectors. A number of bacterial strains includingSalmonella, BCG and Listeria monocytogenes (LM) (Hoiseth & Stocker,Nature 291, 238-239 (1981); Poirier, T P et al. J. Exp. Med. 168, 25-32(1988); (Sadoff, J. C., et al., Science 240, 336-338 (1988); Stover, C.K., et al., Nature 351, 456-460 (1991); Aldovini, A. et al., Nature 351,479-482 (1991); Schafer, R., et al., J. Immunol. 149, 53-59 (1992);Ikonomidis, G. et al., J. Exp. Med. 180, 2209-2218 (1994)).

In addition to virus-mediated gene transfer in vivo, physical meanswell-known in the art can be used for direct transfer of DNA, includingadministration of plasmid DNA (Wolff et al., 1990, supra) andparticle-bombardment mediated gene transfer (Yang, N.-S., et al., Proc.Natl. Acad. Sci. USA 87:9568 (1990); Williams, R. S. et al., Proc. Natl.Acad. Sci. USA 88:2726 (1991); Zelenin, A. V. et al., FEBS Lett. 280:94(1991); Zelenin, A. V. et al., FEBS Lett. 244:65 (1989); Johnston, S. A.et al., In Vitro Cell. Dev. Biol. 27:11 (1991)). Furthermore,electroporation, a well-known means to transfer genes into cell invitro, can be used to transfer DNA molecules to tissues in vivo(Titomirov, A. V. et al., Biochim. Biophys. Acta 1088:131 ((1991)).

“Carrier mediated gene transfer” has also been described (Wu, C. H. etal., J. Biol. Chem. 264:16985 (1989); Wu, G. Y. et al., J. Biol. Chem.263:14621 (1988); Soriano, P. et al., Proc. Natl. Acad. Sci. USA 80:7128(1983); Wang, C-Y. et al., Proc. Natl. Acad. Sci. USA 84:7851 (1982);Wilson, J. M. et al., J. Biol. Chem. 267:963 (1992)). Preferred carriersare targeted liposomes (Nicolau, C. et al., Proc. Natl. Acad. Sci. USA80:1068 (1983); Soriano et al., supra) such as immunoliposomes, whichcan incorporate acylated mAbs into the lipid bilayer (Wang et al.,supra). Polycations such as asialoglycoprotein/polylysine (Wu et al.,1989, supra) may be used, where the conjugate includes a molecule whichrecognizes the target tissue (e.g., asialoorosomucoid for liver) and aDNA binding compound to bind to the DNA to be transfected. Polylysine isan example of a DNA binding molecule which binds DNA without damagingit. This conjugate is then complexed with plasmid DNA.

Plasmid DNA used for transfection or microinjection may be preparedusing methods well-known in the art, for example using the Quiagenprocedure (Quiagen), followed by DNA purification using known methods,such as the methods exemplified herein.

E. Dosages

For LIGHT-HVEM antagonists and nucleic acids encoding LIGHT-HVEMantagonists, as further studies are conducted, information will emergeregarding appropriate dosage levels for treatment of various conditionsin various patients, and the ordinary skilled worker, considering thetherapeutic context, age, and general health of the recipient, will beable to ascertain proper dosing. The selected dosage depends upon thedesired therapeutic effect, on the route of administration, and on theduration of the treatment desired. Generally dosage levels of 0.001 to10 mg/kg of body weight daily are administered to mammals. Generally,for intravenous injection or infusion, dosage may be lower.

EXAMPLES

The present invention may be further understood by reference to thefollowing non-limiting examples.

Example 1 Materials and Methods

Mice:

Female C57BL/6J (B6, H-2^(b)), BALB/c (H-2^(d)), and F1 (B6×DBA/2J)(BDF1; H-2^(b×d)) mice were purchased from the National Cancer Institute(Frederick, Md.). C3H.SW mice (C3.SW-H2^(b)/SnJ) were purchased from TheJackson Laboratory (Bar Harbor, Me.). B6-background LIGHT-KO mice weregenerated in Lieping Chen's laboratory. HVEM-KO mice (H-2^(b)) and 2CTCR transgenic mice were kindly provided by, respectively, Dr. WayneHancock and Dr. Larry Pease (Department of Immunology, Mayo ClinicCollege of Medicine, Rochester, Minn.). Age- and sex-matched 6- to8-week-old mice were used for all experiments. All the animalexperiments described in this manuscript were approved by the AnimalCare and Use Committee of the Johns Hopkins University School ofMedicine.

Cell Lines and Antibodies:

P815 mouse mastocytoma cells (DBA/2, H-2^(d)) and EL4 mouse T-celllymphoma cells (B6, H-2^(b)) were purchased from the American TypeCulture Collection (Rockville, Md.). All cell lines were maintained inthe complete medium under appropriate conditions. Anti-mouse HVEM mAbs(clone; LBH1) were generated by standard techniques as follows. First,Armenian hamsters were immunized subcutaneously (s.c.) andintraperitoneally (i.p.) with 50 μg mouse HVEM-human Fc fusion proteinsemulsified with complete Freund's adjuvant (CFA). Fourteen and 28 dayslater, the hamsters were immunized s.c. and i.p. again with 50 μg mouseHVEM-human Fc fusion protein emulsified with incomplete Freund'sadjuvant (IFA). Thirty five days after the initial immunization, thehamsters were injected intravenously with 50 μg mouse HVEM-human Fc, andsacrified 3 days later. Spleen cells from the hamsters were chemicallyfused with SP2/0 myeloma cells and the hybridomas producing anti-mouseHVEM mAbs were generated by limiting dilution. Control hamster IgG waspurchased from Rockland Immunochemicals (Gilbertsville, Pa.). Anti-2CTCR clonotypic mAb was purified from the supernatants of 1B2 hybridomaand further conjugated with phycoerythrin.

Mouse Parent-to-F1 Transfer GVHD Model in Nonirradiated Hosts:

In the non-irradiated parent-to-F1 GVHD model, 5×10⁷ spleen cellsisolated from wild-type (WT) B6 mice, LIGHT-KO mice, or HVEM-KO micewere transferred intravenously into BDF1 recipients on day 0. In someexperiments, donor spleen cells were labeled with 5 μMcarboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes,Eugene, Oreg.) prior to transfer. In the mice transferred with WT B6splenocytes, 100 μg anti-HVEM mAb or control hamster IgG wasadministered intraperitoneally on days 0, 3, and 6. The recipient micewere killed on the indicated days, and the spleen cells were analyzed byflow cytometry and chromium 51 (⁵¹Cr)-release assay. In the modelemploying 2C T cells, 1×10⁷ spleen cells from 2C TCR-transgenic micewere mixed with 3×10⁷ B6 spleen cells and then transferred intravenouslyinto the BDF1 host on day 0. Recipient mice were subsequentlyadministered 100 μg anti-HVEM mAb or control hamster IgGintraperitoneally on days 0 and 4. On day 7, recipient spleen cells wereharvested and assessed for the presence of 2C T cells by flow cytometricanalysis using 1B2 clonotypic mAb and anti-CD8 mAb.

Mouse GVHD Models Employing Allogeneic BM Transfer Into IrradiatedRecipients:

Three models of GVHD induced by allogeneic bone marrow (BM)transplantation were employed in these studies. First, BDF1 recipientmice, which were preconditioned with lethal irradiation (12 Gy), wereinjected intravenously with T cell-depleted B6 BM cells (5×10⁶ cells)with or without B6 T cells (2-3×10⁶ cells) isolated from either WT orLIGHT-KO mice. T-cell depletion from BM cells and T-cell isolation fromspleen cells was performed by MACS systems using anti-Thy1.2mAb-conjugated microbeads and pan-T cell isolation kits, respectively(Miltenyi Biotec, Auburn, Calif.). In mice transferred with WT B6 Tcells, cohorts of mice were intraperitoneally administered 150 μganti-HVEM mAb or control hamster IgG on days 0, 3, and 6. The survivalof recipient mice was monitored daily. In the second model, BALB/c micewere exposed to lethal irradiation (10 Gy) followed by intravenoustransfer of T cell-depleted B6 BM cells (5×10⁶ cells) with or without B6T cells (1×10⁶ cells) isolated from WT, LIGHT-KO, or HVEM-KO mice. Inthis filly major histocompatibility complex (MHC)-mismatched GVHD model,the survival and body weight change of recipient mice were monitoredregularly.

The third GVHD model was induced by MHC-matched, minorhistocompatibility antigen (miHA)-mismatched BM transfer, B6 mice wereexposed to lethal irradiation (10 Gy) and subsequently injectedintravenously with 4×10⁶ T cell-depleted BM cells from C3H.SW mice(H-2^(b), Ly9.1⁺) with or without 3×10⁷ C3H.SW spleen cells. B6recipient mice injected with C3H.SW spleen cells were intraperitoneallyadministered either anti-HVEM mAb or control hamster IgG at 100 μg ondays 0, 5, 10, 15, 20, and 25. Recipient mice were monitored forsurvival daily and evaluated for body weight and GVHD clinical scoreregularly. For scoring, 5 clinical parameters—weight loss, posture,activity, fur texture, and skin integrity (0-2 in each parameter,maximal score of 10)—were used. In the recipient mice that survived longterm, reconstitution of host lymphoid tissues by donor cells wasassessed by flow cytometry using double staining with Ly9.1 and CD3 orB220. On day 60, cohorts of recipients were killed, and tissues fromliver, skin, and intestine were harvested for pathological analysis byhematoxylin and eosin (H&E) staining. Tissue images were observed usingan Olympus CH30 microscope (Olympus, Center Valley, Pa.) equipped with a20×/0.40 numerical aperture (NA) or a 40×/0.65 NA objective lens. Imageswere acquired using an Olympus DP12 camera and associated imageacquisition software, and were processed using Adobe Photoshop CS2(Adobe Systems, San Jose, Calif.).

Assessment of Division, Apoptosis, and Antihost CTL Activity of DonorCells:

In the GVHD model of parent-to-F1 transfer, division of donor T cellswas assessed by CFSE intensity of H-2K-negative, CD4⁺, or CD8⁺ cells inthe spleen at the indicated time points. Apoptosis of donor T cells wasexamined by Annexin V staining of H-2Kd-negative, CD4⁺, or CD8⁺ spleencells at the indicated time points. Donor anti-host CTL activity wasexamined. Briefly, recipient spleen cells were harvested 10 days afterdonor cell transfer and, without any in vitro manipulation, wereexamined for CTL activity against P815 (H-2^(d)) or EL4 (H-2^(b)) bystandard 4-hour ⁵¹Cr-release assay.

Statistical Analysis:

For survival data, Kaplan-Meier survival curves were prepared usingStatView 5.0 software (SAS Institute, Cary, N.C.), and statisticaldifferences were analyzed using the log-rank (Mantel-Cox) test. P valuesless than 0.05 were considered significant.

Example 2 Indispensable Role of Donor T Cell-Derived LIGHT inGraft-Versus-Host Disease (GVHD) Pathogenesis

To selectively investigate LIGHT functions in GVHD, mice deficient inthe Light gene (LIGHT-KO) were employed. Profound activity of donoranti-host MHC Ag (H-2^(d))-specific CTLs was generated 10 days aftertransfer of wild-type (WT) B6 mice splenocytes into BDF1 mice (FIG. 1A).In sharp contrast, anti-host CTL activity was completely diminished whenLIGHT-KO B6 spleen cells were transferred into BDF1 hosts, indicating acrucial role of donor-derived LIGHT in allo-CTL generation in vivo.LIGHT is expressed and functions on both antigen-presenting cells (APCs)and activated T cells. Therefore, it was important to examine whichsubset among transferred donor cells is responsible for the effects ofLIGHT. To this end, donor cells composed of WT or LIGHT-KO T cellscombined with WT or LIGHT-KO non-T cells were subsequently transferredinto BDF1 mice. Anti-host CTL activity was completely abrogated whenLIGHT-KO T cells were transferred, irrespective of genotypes ofcoinjected non-T cells (FIG. 1B). In contrast, the mice transferred withWT T cells plus LIGHT-KO non-T cells showed a marginal decrease of CTLactivity compared to those injected with WT T and non-T cells. Theseresults strongly indicate that LIGHT associated with donor T cells,rather than APCs, plays an indispensable role in the generation ofanti-host CTLs in vivo.

This notion was bolstered by GVHD models induced by allogeneic BM plusT-cell transfer to lethally irradiated recipient mice. First, BDF1 miceexposed to a lethal dose of irradiation were injected with Tcell-depleted B6 BM cells, together with either WT or LIGHT-KO B6 Tcells. Mice transferred with WT T cells underwent GVHD, and 60% of themdied within 70 days, whereas all mice that underwent transfer withLIGHT-KO T cells survived indefinitely (FIG. 1C). In the second model,fully MHC-mismatched BM transfer was employed as a condition of severeGVHD, in which lethally irradiated BALB/c mice were transferred with Tcell-depleted B6 BM cells plus either WT or LIGHT-KO B6 T cells.Recipient mice transferred with WT T cells all died within 11 days ofsevere GVHD along with profound weight loss (FIG. 1D). In contrast,transfer of LIGHT-KO T cells resulted in a significantly prolongedrecipient survival along with a transient recovery of body weightfollowing acute collapse by the irradiation and BM transfer. Together,these findings indicate an indispensable role of donor T cell-derivedLIGHT in GVHD pathogenesis.

Example 3 Impaired Survival of LIGHT-Deficient Donor T Cells

The cellular mechanisms of the abrogated anti-host CTL activity inLIGHT-KO donor cells was then investigated. The fate of donor T cellsfollowing a transfer into BDF1 recipient mice was monitored. Aftertransfer, the percentage and absolute number of LIGHT-KO donor T cellsin the recipient spleen were significantly lower than those of WT donorT cells (FIG. 2). The decrease of LIGHT-KO donor T cells was moreprominent in CD8⁺ T cells than CD4⁺ T cells. Donor T-cell decrease wasobserved in both hepatic and splenic lymphocytes, suggesting thatchanges of cellular distribution are not responsible for this finding.

The decrease of LIGHT-KO donor T cells could be explained by twopotential mechanisms: an impairment in proliferation, or an accelerationof cell death. In order to address these possibilities, the expansionkinetics of WT and LIGHT-KO donor T cells in vivo was compared. Two tosix days after transfer, division of donor T cells labeled with CFSE wascomparable between WT and LIGHT-KO cells in both CD4⁺ and CD8⁺ T cells(FIG. 3A). This result indicates a dispensable role of LIGHT in drivingthe expansion of alloreactive T cells, thus putting into doubt the firstpossibility. Next, apoptotic cell death in the transferred donor T cellswas investigated. In both spleen and liver, the percentage of AnnexinV-positive cells in LIGHT-KO donor T cells was significantly increasedcompared to those of WT T cells (FIG. 3B). Taken together, thesefindings suggest that deficiency of LIGHT costimulation impairs survivalof host-reactive donor T cells by rendering them vulnerable toactivation-induced cell death.

Example 4 Essential Role of HVEM on Donor T Cells in Their Survival

Among the two functional receptors of LIGHT, HVEM but not LTβR isexpressed on T cells and suggested to be responsible for the T cellcostimulatory effects of LIGHT. In order to directly address a role ofHVEM in GVHD, HVEM-KO lymphocytes were employed as donor cells andtransferred into BDF1 recipient mice. No anti-host CTL activity wasgenerated in the mice injected with HVEM-KO cells, in striking contrastto the ample CTL activity induced by a transfer of control lymphocytes(FIG. 4A). Considering the expression and function of HVEM on broadimmune populations, including DC, T, and B cells, the functional role ofHVEM on donor T and non-T cells was examined using experiments similarto those described in Example 1. Anti-host CTL activity was completelyabrogated when HVEM-KO T cells were transferred as donor cells,irrespective of the genotypes of cotransferred non-T cells, whereas alack of HVEM on non-T cells did not hamper CTL generation whencotransferred with WT T cells (FIG. 4B). These findings indicate thatHVEM on donor T cells plays a crucial role in the generation ofanti-host CTL in GVHD.

HVEM-KO donor T cells undergo massive apoptosis after transfer into therecipient mice and result in a significant decrease of surviving donor Tcells (FIG. 4C). These results concur with the findings in LIGHT-KOdonor cells, suggesting that HVEM is a receptor responsible for LIGHTeffects on donor T-cell survival. The severity of GVHD when HVEM-KOdonor T cells are employed in the fully MHC-mismatched BM transfer modelwas also evaluated. Survival of recipient mice transferred with HVEM-KOcells was significantly prolonged compared to those injected with WTcells (FIG. 4D), highlighting an essential role of donor-derived HVEM inGVHD pathogenesis.

Example 5 Immunotherapy of GVHD by Antagonistic Anti-HVEM mAb

To validate the effects observed in LIGHT-KO or HVEM-KO mice and furtherextend the findings to the treatment of GVHD with a potentialapplication in the clinical setting, anti-HVEM mAbs interfering withLIGHT-HVEM interactions were developed. One of these mAbs, designatedLBH1 was used for further studies. LBH1 abrogates LIGHT-HVEMinteractions while it does not deliver a costimutatory signal when usedin immobilized form (FIGS. 5A and 5B), indicating that LBH1 is anantagonistic mAb. Therapeutic efficacy of LBH1 on GVHD was investigatedby two allogeneic BM transfer models. First, lethally irradiated BDF1mice were injected with T cell-depleted BM cells together with T cellsfrom B6 mice and subsequently were treated with either LBH1 or controlIgG. In this MHC-mismatched model, recipient mice treated with controlIgG succumbed to GVHD by day 75, whereas 40% of the mice treated withLBH1 survived more than 200 days (FIG. 6A). In the second model, GVHDwas induced by MHC-matched, miHA-mismatched BM transfer. Lethallyirradiated B6 mice were injected with T cell-depleted BM cells plus Tcells from C3H.SW mice and were further treated with either control IgGor LBH1. In contrast to less than 30% survival in the recipient micetreated with control IgG, all the mice treated with LBH1 survived morethan 100 days (FIG. 6B). LBH1-treated mice showed significantly lessbody weight loss and improved systemic GVHD scores compared with thosetreated with control IgG (FIG. 6C). After 60 days of BM transfer,control IgG-treated mice displayed a hunched posture and developedsevere GVH skin lesions associated with alopecia, crusting, and erosionformation, whereas none of the mice treated with LBH1 exhibited thesesymptoms. Histologic analysis revealed massive inflammatory cellinfiltration of the portal tracts and bile duct injury in the livers ofcontrol IgG-treated mice but not those treated with LBH1. Skin of thecontrol IgG-treated mice showed epidermal hyperplasia, thickening of thedermis, loss of hair follicles, and profound cellular infiltration,whereas LBH1 treatment prevented such changes. Further histologicevidence of GVHD was shown by the significant number of apoptotic cellsin the intestinal crypt epithelium seen in recipient mice treated withcontrol IgG but not LBH1. In flow cytometric analysis using Ly9.1, whichis a cellular marker expressed on C3H.SW but not B6 mice, hematopoicticcells in the LBH1-treated mice were almost completely replaced by donorcells, indicating an accelerated donor hematopoictic chimerism by thistherapy. In addition, there was no anti-host CTL activity detected inthese long-term surviving mice after LBH1 treatment. Taken together,these results suggest that blockade of the LIGHT-HVEM pathway byantagonistic anti-HVEM mAb effectively ameliorates GVHD associated withallogeneic BM transplantation.

The B6 to BDF1 transfer model was used to investigate the immunologicmechanism of LBH1 therapy. Anti-host CTL activity was profoundlyattenuated by the treatments with LBH1 (FIG. 6A). The number of donor Tcells was significantly decreased by LBH1 treatment without impairingtheir division kinetics (FIG. 7B), suggesting that an analogousmechanism found in LIGHT-KO or HVEM-KO donor cells is operating. Nosignificant decrease of the host immune population was detected,indicating that the effects of LBH1 are not ascribed to the nonspecificdepletion capacity of this mAb. Finally, by employing H-2L^(d)-reactive2C TCR-transgenic T cells, the fate of host Ag-specific donor T cellsafter abrogation of the LIGHT-HVEM costimulatory system was directlymonitored. LBH1 treatment of BDF1 recipient mice, which had beentransferred with 2C T cells and WT B6 spleen cells, resulted in asignificant reduction of 2C T cells in the recipient spleen. Thisfinding suggests that impaired survival of host Ag-specific donor Tcells is responsible for the therapeutic effects of LIGHT-HVEMcostimulatory blockade in GVHD.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims

We claim:
 1. A pharmaceutical composition comprising a LIGHT-HVEMantagonist in an amount effective to reduce or inhibit one or moresymptoms associated with graft rejection or graft-versus-host disease ina host, wherein the LIGHT-HVEM antagonist is an antibody to theextracellular portion of HVEM and does not significantly modulate thebinding LTβ to LTβPR.
 2. The pharmaceutical composition of claim 1further comprising an excipient.
 3. The pharmaceutical composition ofclaim 1, wherein the LIGHT-HVEM antagonist reduces or inhibits thebinding of LIGHT to HVEM.
 4. The pharmaceutical composition of claim 1,wherein the antibody is a monoclonal antibody.
 5. The pharmaceuticalcomposition of claim 4, wherein the antibody has the specificity of themonoclonal antibody LBH1 produced by the hybridoma cell line having ATCCDeposit Number PTA-12171.
 6. The pharmaceutical composition of claim 1,wherein the graft is an allograft selected from the group consisting ofheart, kidney, liver, lung, pancreas, heart valve, cornea, eye lens,bone marrow tissue or endothelial tissue.
 7. A method for treating graftrejection or graft-versus-host disease in a host comprisingadministering a pharmaceutical composition comprising a LIGHT-HVEMantagonist in an amount effective to reduce or inhibit one or moresymptoms associated with graft rejection or graft-versus-host disease ina host, wherein the LIGHT-HVEM antagonist is an antibody to theextracellular portion of HVEM and does not significantly modulate thebinding of LTβ to LTβPR.
 8. The method of claim 7, wherein theLIGHT-HVEM antagonist reduces or inhibits the binding of LIGHT to HVEM.9. The method of claim 7, wherein the antibody is a monoclonal antibody.10. The method of claim 9, wherein the antibody has the specificity ofthe monoclonal antibody LBH1 produced by the hybridoma cell line havingATCC Deposit Number PTA-12171.
 11. The method of claim 7, wherein thegraft is an allograft selected from the group consisting of heart,kidney, liver, lung, pancreas, heart valve, cornea, eye lens, bonemarrow tissue or endothelial tissue.